FIELD OF THE INVENTION
[0001] The present invention relates to monoclonal anti-human-GDF-15 antibodies, pharmaceutical
compositions, kits, methods and uses and the cell lines capable of producing the monoclonal
antibodies described herein. The present invention further relates to antibodies to
human GDF-15 capable of inhibiting cancer growth, treating cancer-induced weight loss
and cancer cachexia.
BACKGROUND
[0002] To date, many cancers are still areas of unmet medical needs, and accordingly, means
to more effectively treat cancer, and to treat cancer in a broader range of cancers
are needed.
[0004] Thus, in order to improve the treatment and prognosis of cancers which lead to cancer
cachexia, treatment regimens that target both of these medical conditions are needed.
To date, most of the emerging drugs for treatments of cancer cachexia are drugs that
target cachexia but not the cancer itself (see
Murphy KT and Lynch GS: Update on emerging drugs for cancer cachexia. Expert Opin
Emerg Drugs. 2009 Dec;14 (4) : 619-32.). Only very few drugs are effective against both the cancer and cancer cachexia,
and therefore, complex treatment regimens that combine anti-cancer drugs and anti-cancer
cachexia drugs are oftentimes needed. Accordingly, there is still an unmet medical
need for drugs that can be used to effectively treat both cancer and cancer cachexia
in a broad range of cancers.
[0005] Many types of cancer are known to express growth factors, including factors such
as VEGF, PDGF, TGF-β and GDF-15.
[0006] GDF-15, growth and differentiation factor-15, is a divergent member of the TGF-β
superfamily. It is a protein which is intracellularly expressed as a precursor, subsequently
processed and eventually becomes secreted from the cell into the environment. Both
the active, fully processed (mature) form and the precursor of GDF-15 can be found
outside cells. The precursor covalently binds via its COOH-terminal amino acid sequence
to the extracellular matrix (
Bauskin AR et al., Cancer Research 2005) and thus resides on the exterior of a cell. The active, fully processed (mature)
form of GDF-15 is soluble and is found in blood sera. Thus, the processed form of
GDF-15 may potentially act on any target cell within the body that is connected to
the blood circulation, provided that the potential target cell expresses a receptor
for the soluble GDF-15 ligand.
[0007] During pregnancy, GDF-15 is found under physiological conditions in the placenta.
However, many malignant cancers (especially aggressive brain cancers, melanoma, lung
cancer, gastrointestinal tumors, colon cancer, pancreatic cancer, prostate cancer
and breast cancer (
Mimeault M and Batra SK, J. Cell Physiol 2010)) exhibit increased GDF-15 levels in the tumor as well as in blood serum. Likewise,
correlations have been described between high GDF-15 expression and chemoresistance
(
Huang CY et al., Clin. Cancer Res. 2009) and between high GDF-15 expression and poor prognosis, respectively (
Brown DA et al., Clin. Cancer Res. 2009).
[0008] GDF-15 is expressed in gliomas of different WHO grades as assessed by immunohistochemistry
(
Roth et al., Clin. Cancer Res. 2010). Further, Roth et al. stably expressed short hairpin RNA-expressing DNA constructs
targeting endogenous GDF-15 or control constructs in SMA560 glioma cells. When using
these pre-established stable cell lines, they observed that tumor formation in mice
bearing GDF-15 knockdown SMA560 cells was delayed compared to mice bearing control
constructs.
[0009] Patent application
PCT/EP2013/070127 relates to monoclonal anti-GDF-15 antibodies, in particular to an antibody produced
by the hybridoma cell line B1-23 deposited with the Deutsche Sammlung für Mikroorganismen
und Zellkulturen GmbH (DSMZ) under the accession No. DSM ACC3142 under the Budapest
treaty.
PCT/EP2013/070127 also relates to uses of the anti-GDF-15 antibodies.
[0010] Patent applications
WO 2005/099746 and
WO 2009/021293 relate to an anti-human-GDF-15 antibody (Mab26) capable of antagonizing effects of
human GDF-15 on tumor-induced weight loss
in vivo in mice: In these documents, immunologically compromised mice were administered with
human tumor cells (prostate carcinoma cells DU145) transfected with plasmids overexpressing
human GDF-15. Tumor cells carrying plasmids lacking a GDF-15 sequence served as a
negative control. Those mice expressing xenograft GDF-15 exhibited a tumor-induced
weight loss (clinical term: cachexia) and anorexia. A single intraperitoneal administration
of 1 mg of Mab26 from
WO 2005/099746 resulted in a complete reversal of tumor-induced weight loss.
WO 2005/099746 and
WO 2009/021293 do not disclose effects of an anti-human-GDF-15 antibody on tumor growth. Moreover,
these documents are silent as to whether anti-human-GDF-15 antibodies could lead to
an increase in body weight of the treated mice compared to their body weight before
the onset of cachexia.
[0011] Similarly,
Johnen H et al. (Nature Medicine, 2007) reported effects of an anti-human-GDF-15 monoclonal antibody on cancer-induced anorexia
and weight loss but did not observe any effects of the anti-human-GDF-15 antibody
on the size of the tumor formed by the cancer, even when the antibody was administered
at a high dosage of 1 mg, and thus the antibody did not inhibit growth of the cancer.
[0012] Accordingly, to date, there was still a need in the art for means to effectively
treat cancer and cancer cachexia, and for means to treat cancer and cancer cachexia
in a broader range of cancers.
[0013] It is therefore an object of the invention to obtain means that can be used to effectively
treat cancer cachexia, and to also effectively treat cancer, and means that can be
used to treat cancer cachexia, and to also effectively treat cancer in a broader range
of cancers.
[0014] In an effort to find means to achieve these objects, the present inventors have surprisingly
found that a monoclonal antibody to human GDF-15 can be used to treat cancer cachexia
and to also treat cancer of human xenograft tumors in mice.
[0015] Additionally, an antibody to human GDF-15 in accordance with the present invention
has an equilibrium dissociation constant of about 790 pM for recombinant GDF-15 even
without additional affinity maturation, which is a higher affinity compared to most
known therapeutic antibodies.
[0016] Thus, the antibody to human GDF-15 according to the present invention has superior
properties compared to antibodies known from the art, and is particularly useful for
inhibiting cancer growth and cancer cachexia. The antibody of the present invention
is therefore useful for treating cancer and for treating cancer cachexia. Accordingly,
the present invention was completed.
BRIEF DESCRIPTION OF THE INVENTION
[0017] The present invention solves the above-mentioned objects by providing the monoclonal
antibodies, pharmaceutical compositions, kits, uses and the cell lines capable of
producing the monoclonal antibodies described herein.
[0018] In particular, the present inventors surprisingly show that monoclonal antibodies
to human GDF-15 and antigen binding portions thereof according to the invention are
capable of inhibiting cancer cachexia and/or cancer growth. This was unexpected because
those monoclonal antibodies to GDF-15 that were previously known from the art (
WO 2005/099746,
WO 2009/021293 and
Johnen H et al., Nature Medicine, 2007) were only known to cause a reversal of cancer-induced weight loss (i.e. a reversal
of a secondary symptom induced by the GDF-15 expressed by the cancer), but were shown
to fail at inhibiting growth of the cancer.
[0019] By showing that the monoclonal antibodies to human GDF-15 according to the invention
can be used to treat cancer-induced weight loss and/or cancer cachexia and treat cancer,
the present inventors also surprisingly show that human GDF-15 protein can be targeted
by the antibodies of the invention in a way that both cancer growth is inhibited and
cancer-induced weight loss and cancer cachexia is treated. It is expected that the
same mechanisms of cancer growth inhibition and treatment of cancer-induced weight
loss and cancer cachexia are applicable to a large number of cancers that overexpress
human GDF-15 including the cancers listed below.
[0020] The monoclonal antibodies and antigen-binding portions thereof according to the invention
are derived from a murine anti-GDF-15 antibody, mAb-B1-23, which was described in
PCT/EP2013/070127 and deposited with the Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH
(DSMZ) under the accession No. DSM ACC3142 under the Budapest treaty. The anti-human
GDF-15 mAb-B1-23 antibodies according to the invention can be generated by replacing
constant domains of the murine antibody mAb-B1-23 with the constant domains of a human
IgG1 antibody.
[0021] Surprisingly, it was observed that a chimeric and a humanized B1-23 antibody according
to the invention showed no tendency to aggregate. These antibody properties according
to the invention are expected to increase the bioavailability of these antibodies
and to be advantageous for clinical formulation of these antibodies.
[0022] Thus, the present invention relates to a monoclonal antibody capable of binding to
human GDF-15, or an antigen-binding portion thereof, wherein the heavy chain variable
domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5
or an amino acid sequence at least 90% identical thereto, and wherein the light chain
variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ
ID NO: 7 or an amino acid sequence at least 85% identical thereto wherein the constant
domain of the heavy chain comprises the amino acid sequence of SEQ ID No: 29, or an
amino acid sequence at least 85%, preferably at least 90%, more preferably at least
95% identical thereto, and wherein the constant domain of the light chain comprises
the amino acid sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95% identical thereto.
[0023] The present invention also relates to a monoclonal antibody capable of binding to
human GDF-15, or an antigen-binding portion thereof, wherein the heavy chain variable
domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5
or an amino acid sequence at least 90% identical thereto, and wherein the light chain
variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ
ID NO: 7 or an amino acid sequence at least 85% identical thereto, for use in a method
for treating cancer cachexia in a mammal. The method comprises administering the antibody
or antigen-binding portion thereof to said mammal. Additionally, the present invention
relates to a corresponding method for treatment.
[0024] Further, the invention also relates to a monoclonal antibody capable of binding to
human GDF-15, or an antigen-binding portion thereof, wherein the binding is binding
to a conformational or discontinuous epitope on human GDF-15 comprised by the amino
acid sequences of SEQ ID No: 25 and SEQ ID No: 26, wherein the constant domain of
the heavy chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid
sequence at least 85%, preferably at least 90%, more preferably at least 95% identical
thereto, and wherein the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95% identical thereto.
[0025] Further, the invention also relates to a monoclonal antibody capable of binding to
human GDF-15, or an antigen-binding portion thereof, wherein the binding is binding
to a conformational or discontinuous epitope on human GDF-15 comprised by the amino
acid sequences of SEQ ID No: 25 and SEQ ID No: 26, for use in a method for treating
cancer cachexia in a mammal. The method comprises administering the antibody or antigen-binding
portion thereof to said mammal. Additionally, the present invention relates to a corresponding
method for treatment.
[0026] The invention also relates to a pharmaceutical composition comprising the antibody
or antigen-binding portion thereof according to the invention.
[0027] The invention also relates to an antibody or antigen-binding portion thereof according
to the invention for use in medicine.
[0028] Further, the invention relates to an antibody or antigen-binding portion thereof
or a pharmaceutical composition according to the invention for use in a method for
treating cancer in a mammal. The method comprises administering the antibody or antigen-binding
portion thereof or the pharmaceutical composition to said mammal.
[0029] Further, the invention relates to an antibody or antigen-binding portion thereof
or a pharmaceutical composition according to the invention for use in a method for
treating cancer cachexia in a mammal. The method comprises administering the antibody
or antigen-binding portion thereof or the pharmaceutical composition to said mammal.
[0030] Additionally, the invention relates to a kit comprising the pharmaceutical composition
according to the invention.
[0031] The invention also relates to an expression vector comprising a nucleotide sequence
encoding the antibody or antigen-binding portion thereof according to the invention.
[0032] Further, the invention relates to a cell line capable of producing an antibody or
antigen-binding portion thereof according to the invention.
[0033] Thus, by providing monoclonal antibodies to human GDF-15, the present invention provides
means for the treatment of cancer cachexia and a cancer growth inhibitor that meets
the above-defined needs in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034]
Figure 1: NKG2D Expression on NK Cells after Treatment with or without GDF-15. The
cell surface expression of NKG2D was determined on NK cells after treatment with the
indicated cytokines in the presence or absence of the anti-GDF-15 antibody mAb Bl-23.
The figure displays specific fluorescence intensities determined by flow cytometry,
quantified relative to an unspecific control antibody.
Figure 2: Akt Phosphorylation in the Ovarian Carcinoma Cell Line SK-OV-3. In order
to quantify the Western Blot for the ovarian carcinoma cell line SK-OV-3, the ratio
of phosphorylated Akt to the total amount of Akt was calculated and normalized to
the untreated control.
Figure 3: JNK1/2 Phosphorylation in Immune Cells. In order to quantify the Western
Blot, the ratio of phosphorylated JNK1/2 to the total amount of JNK was calculated
and normalized to the untreated control.
Figure 4:
An anti-tumor effect of murine B1-23 in vivo. Balb/cnu/nu nude mice were used in a xenograft setting with the melanoma cell line UACC-257.
The tumor size of the animal cohort treated with Bl-23 (open squares) was significantly
decreased, compared to the PBS control group (filled solid circles). Significance
was defined as p<0.05 as assessed by Wilcoxon's log-rank test.
Figure 5: Treatment of cancer cachexia with anti-GDF-15 antibodies. The figure shows
a comparison of the mean body weight of all treated Balb/cnu/nu nude mice, which were inoculated with UACC-257 cells. The changes of the body weight
are depicted in percent as compared to the starting body weight on day 0, for a period
of 38 days.
Figure 6: Coomassie stain of antibodies used in the study No. 140123.
Figure 7: Improved solubility of the chimeric and the humanized antibody at physiological
pH.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0035] Unless otherwise defined below, the terms used in the present invention shall be
understood in accordance with their common meaning known to the person skilled in
the art.
[0036] The term "antibody" as used herein refers to any functional antibody that is capable
of specific binding to the antigen of interest, as generally outlined in
chapter 7 of Paul, W.E. (Ed.).: Fundamental Immunology 2nd Ed. Raven Press, Ltd.,
New York 1989, which is incorporated herein by reference. Without particular limitation, the term
"antibody" encompasses antibodies from any appropriate source species, including chicken
and mammalian such as mouse, goat, non-human primate and human. Preferably, the antibody
is a humanized antibody. The antibody is preferably a monoclonal antibody which can
be prepared by methods well-known in the art. The term "antibody" encompasses an IgG-1,
-2, -3, or -4, IgE, IgA, IgM, or IgD isotype antibody. The term "antibody" encompasses
monomeric antibodies (such as IgD, IgE, IgG) or oligomeric antibodies (such as IgA
or IgM). The term "antibody" also encompasses - without particular limitations - isolated
antibodies and modified antibodies such as genetically engineered antibodies, e.g.
chimeric antibodies.
[0037] The nomenclature of the domains of antibodies follows the terms as known in the art.
Each monomer of an antibody comprises two heavy chains and two light chains, as generally
known in the art. Of these, each heavy and light chain comprises a variable domain
(termed V
H for the heavy chain and V
L for the light chain) which is important for antigen binding. These heavy and light
chain variable domains comprise (in an N-terminal to C-terminal order) the regions
FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 (FR, framework region; CDR, complementarity
determining region which is also known as hypervariable region). The identification
and assignment of the above-mentioned antibody regions within the antibody sequence
is generally in accordance with
Kabat et al. (Sequences of proteins of immunological interest, U.S. Dept. of Health
and Human Services, Public Health Service, National Institutes of Health, Bethesda,
Md. 1983), or
Chothia et al. (Conformations of immunoglobulin hypervariable regions. Nature. 1989
Dec 21-28 ; 342(6252) : 877-83.), or may be performed by using the IMGT/V-QUEST software described in
Giudicelli et al. (IMGT/V-QUEST, an integrated software program for immunoglobulin
and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acids Res. 2004
Jul 1;32(Web Server issue) :W435-40.), which is incorporated herein by reference. Preferably, the antibody regions indicated
above are identified and assigned by using the IMGT/V-QUEST software.
[0038] A "monoclonal antibody" is an antibody from an essentially homogenous population
of antibodies, wherein the antibodies are substantially identical in sequence (i.e.
identical except for minor fraction of antibodies containing naturally occurring sequence
modifications such as amino acid modifications at their N- and C-termini). Unlike
polyclonal antibodies which contain a mixture of different antibodies directed to
numerous epitopes, monoclonal antibodies are directed to the same epitope and are
therefore highly specific. The term "monoclonal antibody" includes (but is not limited
to) antibodies which are obtained from a monoclonal cell population derived from a
single cell clone, as for instance the antibodies generated by the hybridoma method
described in
Köhler and Milstein (Nature, 1975 Aug 7;256(5517):495-7) or
Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York 1988). A monoclonal antibody may also be obtained from other suitable methods, including
phage display techniques such as those described in
Clackson et al. (Nature. 1991 Aug 15 ; 352 (6336): 624-8) or
Marks et al. (J Mol Biol. 1991 Dec 5 ; 222(3) : 581-97). A monoclonal antibody may be an antibody that has been optimized for antigen-binding
properties such as decreased Kd values, optimized association and dissociation kinetics
by methods known in the art. For instance, Kd values may be optimized by display methods
including phage display, resulting in affinity-matured monoclonal antibodies. The
term "monoclonal antibody" is not limited to antibody sequences from particular species
of origin or from one single species of origin. Thus, the meaning of the term "monoclonal
antibody" encompasses chimeric monoclonal antibodies such as humanized monoclonal
antibodies.
[0039] "Humanized antibodies" are antibodies which contain human sequences and a minor portion
of non-human sequences which confer binding specificity to an antigen of interest
(e.g. human GDF-15). Typically, humanized antibodies are generated by replacing hypervariable
region sequences from a human acceptor antibody by hypervariable region sequences
from a non-human donor antibody (e.g. a mouse, rabbit, rat donor antibody) that binds
to an antigen of interest (e.g. human GDF-15). In some cases, framework region sequences
of the acceptor antibody may also be replaced by the corresponding sequences of the
donor antibody. In addition to the sequences derived from the donor and acceptor antibodies,
a "humanized antibody" may either contain other (additional or substitute) residues
or sequences or not. Such other residues or sequences may serve to further improve
antibody properties such as binding properties (e.g. to decrease Kd values) and/or
immunogenic properties (e.g. to decrease antigenicity in humans). Non-limiting examples
for methods to generate humanized antibodies are known in the art, e.g. from
Riechmann et al. (Nature. 1988 Mar 24 ; 332(6162) : 323-7) or
Jones et al. (Nature. 1986 May 29-Jun 4;321(6069):522-5).
[0040] The term "human antibody" relates to an antibody containing human variable and constant
domain sequences. This definition encompasses antibodies having human sequences bearing
single amino acid substitutions or modifications which may serve to further improve
antibody properties such as binding properties (e.g. to decrease Kd values) and/or
immunogenic properties (e.g. to decrease antigenicity in humans). The term "human
antibody" excludes humanized antibodies where a portion of non-human sequences confers
binding specificity to an antigen of interest.
[0041] An "antigen-binding portion" of an antibody as used herein refers to a portion of
an antibody that retains the capability of the antibody to specifically bind to the
antigen (e.g. GDF-15), i.e. the "antigen-binding portion" is capable of competing
with the antibody for specific binding to the antigen. The "antigen-binding portion"
may contain one or more fragments of the antibody. Without particular limitation,
it can be produced by any suitable method known in the art, including recombinant
DNA methods and preparation by chemical or enzymatic fragmentation of antibodies.
Antigen-binding portions may be Fab fragments, F(ab') fragments, F(ab')
2 fragments, single chain antibodies (scFv), single-domain antibodies, diabodies or
any other portion(s) of the antibody that allow (s) to retain binding to the antigen.
[0042] An "antibody" (e.g. a monoclonal antibody) or an "antigen-binding portion" may have
been derivatized or be linked to a different molecule. For example, molecules that
may be linked to the antibody are other proteins (e.g. other antibodies), a molecular
label (e.g. a fluorescent, luminescent, colored or radioactive molecule), a pharmaceutical
and/or a toxic agent. The antibody or antigen-binding portion may be linked directly
(e.g. in form of a fusion between two proteins), or via a linker molecule (e.g. any
suitable type of chemical linker known in the art).
[0043] As used herein, the terms "binding" or "bind" refer to specific binding to the antigen
of interest (e.g. human GDF-15) . Preferably, the Kd value is less than 100 nM, more
preferably less than 50 nM, still more preferably less than 10 nM, still more preferably
less than 5 nM and most preferably less than 2 nM.
[0044] The term "epitope" as used herein refers to a small portion of an antigen that forms
the binding site for an antibody.
[0045] In the context of the present invention, binding or competitive binding of antibodies
or their antigen-binding portions to the antigen of interest (e.g. human GDF-15) is
measured by using surface plasmon resonance measurements as a reference standard assay,
as described below.
[0046] The terms "K
D" or "K
D value" relate to the equilibrium dissociation constant as known in the art. In the
context of the present invention, these terms relate to the equilibrium dissociation
constant of an antibody with respect to a particular antigen of interest (e.g. human
GDF-15). The equilibrium dissociation constant is a measure of the propensity of a
complex (e.g. an antigen-antibody complex) to reversibly dissociate into its components
(e.g. the antigen and the antibody). For the antibodies according to the invention,
K
D values (such as those for the antigen human GDF-15) are generally determined by using
surface plasmon resonance measurements as described below.
[0047] The term "cancer growth" as used herein relates to any measureable growth of the
cancer. For cancers forming solid tumors, "cancer growth" relates to a measurable
increase in tumor volume over time. If the cancer has formed only a single tumor,
"cancer growth" relates only to the increase in volume of the single tumor. If the
cancer has formed multiple tumors such as metastases, "cancer growth" relates to the
increase in volume of all measurable tumors. For solid tumors, the tumor volume can
be measured by any method known in the art, including magnetic resonance imaging and
computed tomography (CT scan).
[0048] For leukemias which are characterized by the presence of cancerous cells of the blood
system in blood, "cancer growth" relates to a measurable increase in the number of
cancer cells per blood volume. In order to carry out such measurements, cancer cells
can be identified from blood samples by using any method known in the art, including
cell morphology measurements, or staining of tumor cell marker proteins such as tumor
marker cell surface proteins, e.g. by staining with specific antibodies, and the cancer
cells can be counted.
[0049] Terms such as "inhibiting cancer growth" as used herein refer to a measurable inhibition
of cancer growth in patient treated with the antibody. Preferably, the inhibition
is statistically significant. Inhibition of cancer growth may be assessed by comparing
cancer growth in a group of patients treated in accordance with the present invention
to a control group of untreated patients, or by comparing a group of patients that
receive a standard cancer treatment of the art plus a treatment according to the invention
with a control group of patients that only receive a standard cancer treatment of
the art. Such studies for assessing the inhibition of cancer growth are designed in
accordance with accepted standards for clinical studies, e.g. double-blinded, randomized
studies with sufficient statistical power. The term "inhibiting cancer growth" includes
an inhibition of cancer growth where the cancer growth is inhibited partially (i.e.
where the cancer growth in the patient is delayed compared to the control group of
patients), an inhibition where the cancer growth is inhibited completely (i.e. where
the cancer growth in the patient is stopped), and an inhibition where cancer growth
is reversed (i.e. the cancer shrinks).
[0050] An "isolated antibody" as used herein is an antibody that has been identified and
separated from the majority of components (by weight) of its source environment, e.g.
from the components of a hybridoma cell culture or a different cell culture that was
used for its production (e.g. producer cells such as CHO cells that recombinantly
express the antibody). The separation is performed such that it sufficiently removes
components that may otherwise interfere with the suitability of the antibody for the
desired applications (e.g. with a therapeutic use of the anti-human GDF-15 antibody
according to the invention). Methods for preparing isolated antibodies are known in
the art and include Protein A chromatography, anion exchange chromatography, cation
exchange chromatography, virus retentive filtration and ultrafiltration. Preferably,
the isolated antibody preparation is at least 70 % pure (w/w), more preferably at
least 80 % pure (w/w), still more preferably at least 90 % pure (w/w), still more
preferably at least 95 % pure (w/w), and most preferably at least 99 % pure (w/w),
as measured by using the Lowry protein assay.
[0051] A "diabody" as used herein is a small bivalent antigen-binding antibody portion which
comprises a heavy chain variable domain linked to a light chain variable domain on
the same polypeptide chain linked by a peptide linker that is too short to allow pairing
between the two domains on the same chain. This results in pairing with the complementary
domains of another chain and in the assembly of a dimeric molecule with two antigen
binding sites. Diabodies may be bivalent and monospecific (such as diabodies with
two antigen binding sites for human GDF-15), or may be bivalent and bispecific (e.g.
diabodies with two antigen binding sites, one being a binding site for human GDF-15,
and the other one being a binding site for a different antigen). A detailed description
of diabodies can be found in
Holliger P et al. (""Diabodies": small bivalent and bispecific antibody fragments."
Proc Natl Acad Sci USA. 1993 Jul 15 ; 90 (14) : 6444-8.).
[0053] The term "higher" as used herein means that a value (e.g. a GDF-15 level) in a patient
sample is higher than a value in a corresponding control sample or group of control
samples. Preferably, the difference is statistically significant.
[0054] The term "elevated GDF-15 levels" as used herein means that the human patient has
higher GDF-15 levels in blood serum before administration of the antibody or antigen-binding
portion thereof or the pharmaceutical composition according to the invention, when
compared to median GDF-15 levels in blood sera of healthy human control individuals
as a reference.
[0056] The term "prior to administration" as used herein means the period of time immediately
before administration of the antibody, fragment thereof or the pharmaceutical composition
according to the invention. Preferably, the term "prior to administration" means a
period of 30 days immediately before administration; most preferably a period of one
week immediately before administration.
[0057] The terms "significant", "significantly", etc. as used herein refer to a statistically
significant difference between values.
[0059] The term "cancer-induced weight loss" is used herein in accordance with its common
meaning in the art. Cancer-induced weight loss is frequently seen as an adverse effect
in individuals having cancer (see, for instance
Fearon K. et al.: Definition and classification of cancer cachexia: an international
consensus. Lancet Oncol. 2011 May; 12(5):489-95.;
Tisdale MJ.: Mechanisms of cancer cachexia. Physiol Rev. 2009 Apr;89(2):381-410.). The term "cancer-induced weight loss" relates to the body weight loss induced
by the cancer. Additional body weight loss in addition to the cancer-induced weight
loss - e.g. body weight loss induced by cancer treatments such as surgery, chemotherapy
and radiotherapy - can also occur in individuals having cancer. It is understood that
the meaning of the term "cancer-induced weight loss" does not include this additional
body weight loss. However, this does not exclude the possibility that the antibodies
of the present invention - in addition to their effects on cancer-induced weight loss
and on cancer growth - may have beneficial effects against such additional body weight
loss, e.g. by reverting or partly reverting such additional weight loss, or by preventing
or partly preventing such additional body weight loss.
[0060] Body weight can easily be measured by weighing, and body weight is typically expressed
in units of mass such as kg.
[0062] With respect to cancer cachexia, a "treatment" according to the present invention
may be a treatment for preventing and/or a treatment for inhibiting or reverting cancer
cachexia. Typically, a treatment for preventing cancer cachexia is a treatment that
is given prophylactically at a stage of the cancer disease where no cancer cachexia
has yet occurred. A treatment for inhibiting cancer cachexia is typically a treatment
that is given at a stage of the cancer disease where some cancer cachexia has occurred,
in order to inhibit a further progression of the cancer cachexia. A treatment for
reverting cancer cachexia is typically a treatment that is started at a stage of the
cancer where some cancer cachexia has occurred, and which reverts the cancer cachexia.
The effect of the treatment can be a partial effect, i.e. a partial prevention, a
partial inhibition or a partial reversion of cancer cachexia, or a complete effect,
i.e. a complete prevention, a complete inhibition or a complete reversion of cancer
cachexia. Preferably, according to the present invention, the effect of the treatment
is a complete prevention, a complete inhibition or a complete reversion of cancer
cachexia. More preferably, the effect of the treatment according to the present invention
is a complete prevention or a complete reversion of cancer cachexia.
[0063] As used herein, the term "complete(ly)" in connection with a treatment of cancer
cachexia according to the invention means that in case of a treatment for preventing,
no cancer cachexia occurs in the treated individual during and/or following the treatment.
In case of a treatment for inhibiting cancer cachexia, the term "complete(ly)" means
that no further progression of the cancer cachexia occurs in the treated individual
during and/or following the treatment. In case of a treatment for reverting cancer
cachexia, the term "complete(ly)" means that during or following the treatment, the
cancer cachexia is completely reverted such that no cancer cachexia is present in
the treated individual.
[0065] In addition to completely preventing or completely reverting cancer cachexia, the
treatment methods and products for use in these methods according to the invention
may increase the body weight of the treated mammal compared to its body weight before
the onset of cancer cachexia. As used herein, the term "before the onset of cancer
cachexia" means a point in time during the course of the cancer disease, after which
cancer cachexia becomes measurable by the methods known in the art such as the methods
referred to above.
[0066] Preferably, the above-defined effects of the cancer cachexia treatment according
to the invention are statistically significant when assessed against a suitable control
group whereas individual patients who are treated would not show significant cachexia.
[0067] In accordance with the present invention, each occurrence of the term "comprising"
may optionally be substituted with the term "consisting of".
Methods and Techniques
[0068] Generally, unless otherwise defined herein, the methods used in the present invention
(e.g. cloning methods or methods relating to antibodies) are performed in accordance
with procedures known in the art, e.g. the procedures described in
Sambrook et al. ("Molecular Cloning: A Laboratory Manual.", 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York 1989),
Ausubel et al. ("Current Protocols in Molecular Biology." Greene Publishing Associates
and Wiley Interscience; New York 1992), and
Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York 1988), all of which are incorporated herein by reference.
[0069] Molecular weight is measured by methods known in the art such as mass spectrometry.
It is expressed in Dalton (Da) or Kilodalton (kDa).
[0070] Binding of monoclonal anti-human-GDF-15 antibodies according to the invention is
generally assessed by employing surface plasmon resonance measurements using a Bio-Rad®
ProteOn™ XPR36 system and Bio-Rad® GLC sensor chips as described for murine anti-human
GDF-15 mAb-B1-23 in Example 1.
[0072] Monoclonal antibodies according to the invention can be produced by any method known
in the art, including but not limited to the methods referred to in
Siegel DL ("Recombinant monoclonal antibody technology." Transfus Clin Biol. 2002
Jan;9(1):15-22.). In a preferred embodiment, an antibody according to the invention is produced
by the hybridoma cell line B1-23 deposited with the Deutsche Sammlung für Mikroorganismen
und Zellkulturen GmbH (DSMZ) under the accession No. DSM ACC3142 under the Budapest
treaty. The deposit was filed on September 29, 2011.
[0073] Cell proliferation can be measured by suitable methods known in the art, including
(but not limited to) visual microscopy, metabolic assays such as those which measure
mitochondrial redox potential (e.g. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay; Resazurin staining which is also known as Alamar Blue® assay), staining
of known endogenous proliferation biomarkers (e.g. Ki-67), and methods measuring cellular
DNA synthesis (e.g. BrdU and [
3H]-Thymidine incorporation assays).
[0074] Immunosuppression can be measured by suitable methods known in the art, including
(but not limited to) immune cell proliferation, cytokine secretion, intracellular
cytokine staining by flow cytometry, cytokine measurement by qRT-PCR, redirected target
cell lysis, further cytotoxicity or degranulation assays, downregulation of activating
immune cell receptors (like NKG2D), upregulation of inhibitory immune cell receptors,
immunological synapse formation, immune cell infiltration. For the term immunosuppression
to apply, an effect shall be measurable in at least one of these or in any other suitable
assay. The lack of effect in a specific test does not imply a general absence of immunosuppression.
[0075] Human GDF-15 levels can be measured by any method known in the art, including measurements
of GDF-15 mRNA levels by methods including (but not limited to) quantitative real-time
PCR (qRT-PCR) for human GDF-15 mRNA using primers specific to human GDF-15, mRNA in
situ hybridization with probes specific to human GDF-15, mRNA deep sequencing methods;
and including measurements of GDF-15 protein levels by methods including (but not
limited to) mass spectrometry for proteins or peptides derived from human GDF-15,
Western Blotting using antibodies specific to human GDF-15, flow cytometry using antibodies
specific to human GDF-15, strip tests using antibodies specific to human GDF-15, or
immunocytochemistry using antibodies specific to human GDF-15. For such methods using
antibodies specific to human GDF-15, the anti-human GDF-15 antibodies of the present
invention are preferred, and the antibody of the invention produced by the hybridoma
cell line B1-23 deposited with the Deutsche Sammlung für Mikroorganismen und Zellkulturen
GmbH (DSMZ) under the accession No. DSM ACC3142 is most preferred.
Embodiments of the Invention
[0076] As described above, the inventors show that human GDF-15 protein can be targeted
by an antibody in accordance with the invention in a way that cancer cachexia and
cancer-induced weight loss can be treated and that also cancer growth is inhibited.
[0077] When taking into account the present invention, it becomes clear that the anti-GDF-15
antibodies known from
WO 2005/099746,
WO 2009/021293 and
Johnen H et al., Nature Medicine, 2007 only inhibit one of the effects of human GDF-15 (i.e. cancer-induced weight loss),
but fail to inhibit other effects of human GDF-15 such as those related to cancer
growth. In view of the present invention, one possible explanation for this failure
is that the antibodies known from the above documents may only interfere with transport
of human GDF-15 across the blood-brain barrier (by forming a large complex that cannot
be transported across the blood-brain barrier) but are incapable of binding human
GDF-15 in a way that renders it generally unable to interact with its receptor (e.g.
a receptor residing on cells outside the brain). Furthermore, and different from the
antibodies of the present invention, the anti-GDF-15 antibodies known from
WO 2005/099746,
WO 2009/021293 and
Johnen H et al., Nature Medicine, 2007 did not lead to a detectable increase in the body weight of the mammals compared
to its body weight before the onset of cancer cachexia.
[0078] Accordingly, the effects of the antibodies for use according to the invention are
unexpected in view of the art.
[0079] The following properties of the antibodies of the present invention are expected
to contribute to their capability of inhibiting the effects of human GDF-15 more completely,
including the treatment of cachexia and the inhibition of cancer growth:
Broad Binding Specificity to Forms of Human GDF-15
[0080] The antibodies of the present invention are capable of binding to mature recombinant
human GDF-15 (represented by SEQ ID No: 8) and are therefore capable of binding to
active, fully processed (mature) human GDF-15.
[0081] Additionally, by performing staining experiments with the murine mAb-B1-23 antibody
according to the invention on human cells, the inventors show that the mAb-B1-23 antibody
according to the invention is capable of binding to the human GDF-15 precursor on
human cells.
[0082] Thus, it is expected that binding and effects of the antibodies according to the
present invention, in particular the inhibition of cancer growth, are not generally
limited to effects on a particular form of human GDF-15.
[0083] As to the effects of human GDF-15 on cancer cachexia, these effects may be caused
a subset of forms human GDF-15, for instance to soluble forms human GDF-15, which
are capable of passing the blood-brain barrier. As exemplified in the Examples of
the present invention, all of the tested anti-GDF-15 antibodies according to the invention
can be used to treat cancer-induced cachexia. Thus, the antibodies according to the
present invention can interfere with the forms of human GDF-15 which are responsible
for cancer cachexia.
High Binding Affinity
[0084] The antibodies and antigen binding portions thereof according to the invention have
high binding affinity, as demonstrated by the mAb-B1-23 antibody according to the
invention which has an equilibrium dissociation constant of about 790pM for recombinant
human GDF-15. Notably, such affinity values are superior to most of the existing therapeutic
antibodies, e.g. to the therapeutic antibody Rituximab which has an equilibrium dissociation
constant of about 8 nM.
[0085] High binding affinity will ensure that the antibody to human GDF-15 according to
the invention stably binds to human GDF-15, such that effects of human GDF-15 including
effects on cancer growth are effectively inhibited. Likewise, stable binding of the
antibodies according to the invention is expected to ensure that forms of human GDF-15
which cause cancer cachexia cannot carry out their pathological function. This may
for instance be due to an antibody-dependent sequestration of these forms of human
GDF-15 from their possible site of action in the brain. Such binding and sequestration
may for instance take place at the site of the cancer, or the antibodies according
to the invention may interfere with the transport of human-GDF-15 across the blood
brain-barrier.
Binding to a Discontinuous or Conformational Epitope
[0086] The antibodies and antigen binding portions thereof according to the invention bind
to a discontinuous or conformational epitope, as demonstrated below for a murine mAb-B1-23
antibody according to the invention.
[0087] Binding of antibodies and antigen binding portions thereof according to the invention
to a discontinuous or conformational GDF-15 epitope may help to keep human GDF-15
in a specific conformation. This conformation-specificity may be advantageous to keep
GDF-15 in a form that cannot be released from the tumor, or that cannot cross the
blood brain-barrier and cause cancer cachexia at a possible site of action in the
brain. Additionally, such binding to a discontinuous or conformational GDF-15 epitope
may contribute to the effective inhibition of effects of human GDF-15 including effects
on cancer growth, e.g. by keeping GDF-15 in a conformation that cannot functionally
interact with its receptor.
[0088] Thus, the invention relates to the following embodiments:
A) Antibodies, Vectors and Cell Lines
[0089] Concretely, the invention relates to a monoclonal antibody capable of binding to
human GDF-15, or an antigen-binding portion thereof, wherein the heavy chain variable
domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5
or an amino acid sequence at least 90% identical thereto, and wherein the light chain
variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ
ID NO: 7 or an amino acid sequence at least 85% identical thereto, wherein the constant
domain of the heavy chain comprises the amino acid sequence of SEQ ID No: 29, or an
amino acid sequence at least 85%, preferably at least 90%, more preferably at least
95% identical thereto, and wherein the constant domain of the light chain comprises
the amino acid sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95% identical thereto.
[0090] In a preferred aspect of this embodiment, the constant domain of the heavy chain
comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence at least
98%, preferably at least 99% identical thereto, and the constant domain of the light
chain comprises the amino acid sequence of SEQ ID No: 32, or an amino acid sequence
at least 98%, preferably at least 99% identical thereto.
[0091] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises an amino acid sequence at least 85% identical to the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95%, still more preferably at least 98%, and most preferably
at least 99% identical thereto.
[0092] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises an amino acid sequence at least 90% identical to the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95%, still more preferably at least 98%, and most preferably
at least 99% identical thereto.
[0093] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises an amino acid sequence at least 95% identical to the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95%, still more preferably at least 98%, and most preferably
at least 99% identical thereto.
[0094] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises an amino acid sequence at least 98% identical to the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95%, still more preferably at least 98%, and most preferably
at least 99% identical thereto.
[0095] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises an amino acid sequence at least 99% identical to the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95%, still more preferably at least 98%, and most preferably
at least 99% identical thereto.
[0096] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably
at least 98%, and most preferably at least 99% identical thereto, and the constant
domain of the light chain comprises an amino acid sequence at least 85% identical
to the amino acid sequence of SEQ ID No: 32.
[0097] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably
at least 98%, and most preferably at least 99% identical thereto, and the constant
domain of the light chain comprises an amino acid sequence at least 90% identical
to the amino acid sequence of SEQ ID No: 32.
[0098] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably
at least 98%, and most preferably at least 99% identical thereto, and the constant
domain of the light chain comprises an amino acid sequence at least 95% identical
to the amino acid sequence of SEQ ID No: 32.
[0099] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably
at least 98%, and most preferably at least 99% identical thereto, and the constant
domain of the light chain comprises an amino acid sequence at least 98% identical
to the amino acid sequence of SEQ ID No: 32.
[0100] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably
at least 98%, and most preferably at least 99% identical thereto, and the constant
domain of the light chain comprises an amino acid sequence at least 99% identical
to the amino acid sequence of SEQ ID No: 32.
[0101] In this embodiment, most preferably, the constant domain of the heavy chain comprises
the amino acid sequence of SEQ ID No: 29, and the constant domain of the light chain
comprises the amino acid sequence of SEQ ID No: 32.
[0102] In an alternative embodiment, the invention relates to a monoclonal antibody capable
of binding to human GDF-15, or an antigen-binding portion thereof, wherein the heavy
chain variable domain comprises a CDR3 region comprising the amino acid sequence of
SEQ ID NO: 5 or an amino acid sequence that differs by not more than one amino acid
from the amino acid sequence of SEQ ID NO: 5, and wherein the light chain variable
domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 7
or an amino acid sequence or an amino acid sequence that differs by not more than
one amino acid from the amino acid sequence of SEQ ID NO: 7, wherein the constant
domain of the heavy chain comprises the amino acid sequence of SEQ ID No: 29, or an
amino acid sequence at least 85%, preferably at least 90%, more preferably at least
95% identical thereto, and wherein the constant domain of the light chain comprises
the amino acid sequence of SEQ ID No: 32, or an amino acid sequence at least 85%,
preferably at least 90%, more preferably at least 95% identical thereto.
[0103] In a preferred aspect of this embodiment, the constant domain of the heavy chain
comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence at least
98%, preferably at least 99% identical thereto, and the constant domain of the light
chain comprises the amino acid sequence of SEQ ID No: 32, or an amino acid sequence
at least 98%, preferably at least 99% identical thereto.
[0104] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises an amino acid sequence at least 85% identical to the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95%, still more preferably at least 98%, and most preferably
at least 99% identical thereto.
[0105] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises an amino acid sequence at least 90% identical to the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95%, still more preferably at least 98%, and most preferably
at least 99% identical thereto.
[0106] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises an amino acid sequence at least 95% identical to the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95%, still more preferably at least 98%, and most preferably
at least 99% identical thereto.
[0107] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises an amino acid sequence at least 98% identical to the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95%, still more preferably at least 98%, and most preferably
at least 99% identical thereto.
[0108] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises an amino acid sequence at least 99% identical to the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least
90%, more preferably at least 95%, still more preferably at least 98%, and most preferably
at least 99% identical thereto.
[0109] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably
at least 98%, and most preferably at least 99% identical thereto, and the constant
domain of the light chain comprises an amino acid sequence at least 85% identical
to the amino acid sequence of SEQ ID No: 32.
[0110] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably
at least 98%, and most preferably at least 99% identical thereto, and the constant
domain of the light chain comprises an amino acid sequence at least 90% identical
to the amino acid sequence of SEQ ID No: 32.
[0111] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably
at least 98%, and most preferably at least 99% identical thereto, and the constant
domain of the light chain comprises an amino acid sequence at least 95% identical
to the amino acid sequence of SEQ ID No: 32.
[0112] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably
at least 98%, and most preferably at least 99% identical thereto, and the constant
domain of the light chain comprises an amino acid sequence at least 98% identical
to the amino acid sequence of SEQ ID No: 32.
[0113] In another preferred aspect of this embodiment, the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably
at least 98%, and most preferably at least 99% identical thereto, and the constant
domain of the light chain comprises an amino acid sequence at least 99% identical
to the amino acid sequence of SEQ ID No: 32.
[0114] In this embodiment, most preferably, the constant domain of the heavy chain comprises
the amino acid sequence of SEQ ID No: 29, and the constant domain of the light chain
comprises the amino acid sequence of SEQ ID No: 32.
[0115] Further, a monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof is provided, wherein the heavy chain variable domain comprises a CDR3
region comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence
at least 90% identical thereto, and wherein the light chain variable domain comprises
a CDR3 region comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid
sequence at least 85% identical thereto. Preferably, the constant domain of the heavy
chain of this monoclonal antibody or antigen-binding portion thereof comprises the
amino acid sequence of SEQ ID No: 29, or an amino acid sequence at least 85%, preferably
at least 90%, more preferably at least 95% identical thereto, and the constant domain
of the light chain of this monoclonal antibody or antigen-binding portion thereof
comprises the amino acid sequence of SEQ ID No: 32, or an amino acid sequence at least
85%, preferably at least 90%, more preferably at least 95% identical thereto. More
preferably, the constant domain of the heavy chain comprises the amino acid sequence
of SEQ ID No: 29, or an amino acid sequence at least 98%, preferably at least 99%
identical thereto, and the constant domain of the light chain comprises the amino
acid sequence of SEQ ID No: 32, or an amino acid sequence at least 98%, preferably
at least 99% identical thereto. Still more preferably, the constant domain of the
heavy chain comprises the amino acid sequence of SEQ ID No: 29, and the constant domain
of the light chain comprises the amino acid sequence of SEQ ID No: 32.
[0116] Further, a monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof is provided, wherein the heavy chain variable domain comprises a CDR3
region comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence
that differs by not more than one amino acid from the amino acid sequence of SEQ ID
NO: 5, and wherein the light chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence or an amino acid
sequence that differs by not more than one amino acid from the amino acid sequence
of SEQ ID NO: 7. Preferably, the constant domain of the heavy chain of this monoclonal
antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ
ID No: 29, or an amino acid sequence at least 85%, preferably at least 90%, more preferably
at least 95% identical thereto, and the constant domain of the light chain of this
monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence
of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least 90%,
more preferably at least 95% identical thereto. More preferably, the constant domain
of the heavy chain comprises the amino acid sequence of SEQ ID No: 29, or an amino
acid sequence at least 98%, preferably at least 99% identical thereto, and the constant
domain of the light chain comprises the amino acid sequence of SEQ ID No: 32, or an
amino acid sequence at least 98%, preferably at least 99% identical thereto. Still
more preferably, the constant domain of the heavy chain comprises the amino acid sequence
of SEQ ID No: 29, and the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32.
[0117] In a second embodiment in accordance with the above embodiments, the heavy chain
variable domain of the monoclonal antibody or antigen-binding portion thereof comprises
a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5, or the light chain
variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ
ID NO: 7.
[0118] In a third embodiment in accordance with the above embodiments, the heavy chain variable
domain of the monoclonal antibody or antigen-binding portion thereof comprises a CDR3
region comprising the amino acid sequence of SEQ ID NO: 5, and the light chain variable
domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 7.
[0119] In a fourth embodiment in accordance with the above embodiments, the heavy chain
variable domain of the monoclonal antibody or antigen-binding portion thereof comprises
a CDR1 region comprising the amino acid sequence of SEQ ID NO: 3 and a CDR2 region
comprising the amino acid sequence of SEQ ID NO: 4, and the light chain variable domain
of the monoclonal antibody or antigen-binding portion thereof comprises a CDR1 region
comprising the amino acid sequence of SEQ ID NO: 6 and a CDR2 region comprising the
amino acid sequence ser-ala-ser.
[0120] In a fifth embodiment in accordance with the above embodiments, the antibody is a
humanized antibody. Preferably, all of the variable domains of the humanized antibody
are humanized variable domains.
[0121] In a further embodiment in accordance with the above embodiments, the heavy chain
variable domain of the monoclonal antibody or antigen-binding portion thereof comprises
the amino acid sequence of SEQ ID No: 28, or an amino acid sequence at least 90%,
preferably at least 95%, more preferably at least 98%, still more preferably at least
99% identical thereto, and the light chain variable domain of the monoclonal antibody
or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID No:
31, or an amino acid sequence at least 90%, preferably at least 95%, more preferably
at least 98%, still more preferably at least 99% identical thereto. In the most preferred
aspect of this embodiment, the heavy chain variable domain comprises the amino acid
sequence of SEQ ID No: 28, and the light chain variable domain comprises the amino
acid sequence of SEQ ID No:31.
[0122] In a further preferred embodiment in accordance with the above embodiments, the heavy
chain of the monoclonal antibody or antigen-binding portion thereof comprises the
amino acid sequence of SEQ ID No: 27, and the light chain of the monoclonal antibody
or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID No:
30.
[0123] In a another preferred embodiment in accordance with the above embodiments, the heavy
chain variable domain of the monoclonal antibody or antigen-binding portion thereof
comprises the amino acid sequence of SEQ ID No: 34, or an amino acid sequence at least
75%, more preferably at least 80%, more preferably at least 85%, more preferably at
least 90%, more preferably at least 95%, more preferably at least 98%, still more
preferably at least 99% identical thereto, and the light chain variable domain of
the monoclonal antibody or antigen-binding portion thereof comprises the amino acid
sequence of SEQ ID No: 37, or an amino acid sequence at least 80%, more preferably
at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably
at least 98%, still more preferably at least 99% identical thereto. In the most preferred
aspect of this embodiment in accordance with the above embodiments, the heavy chain
variable domain comprises the amino acid sequence of SEQ ID No: 34, and the light
chain variable domain comprises the amino acid sequence of SEQ ID No: 37.
[0124] In still another embodiment in accordance with the above first to third embodiment,
the heavy chain variable domain comprises a region comprising an FR1, a CDR1, an FR2,
a CDR2 and an FR3 region and comprising the amino acid sequence of SEQ ID NO: 1 or
a sequence 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto,
and the light chain variable domain comprises a region comprising an FR1, a CDR1,
an FR2, a CDR2 and an FR3 region and comprising the amino acid sequence of SEQ ID
NO: 2 or a sequence 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical
thereto.
[0125] In a preferred embodiment in accordance with the above first to third embodiment,
the heavy chain variable domain comprises a region comprising an FR1, a CDR1, an FR2,
a CDR2 and an FR3 region and comprising the amino acid sequence of SEQ ID NO: 1 or
a sequence 95% identical thereto, and the light chain variable domain comprises a
region comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region and comprising
the amino acid sequence of SEQ ID NO: 2 or a sequence 95% identical thereto.
[0126] In a more preferred embodiment in accordance with the above first to third embodiment,
the heavy chain variable domain comprises a region comprising an FR1, a CDR1, an FR2,
a CDR2 and an FR3 region and comprising the amino acid sequence of SEQ ID NO: 1 or
a sequence 98% identical thereto, and the light chain variable domain comprises a
region comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region and comprising
the amino acid sequence of SEQ ID NO: 2 or a sequence 98% identical thereto.
[0127] In a still more preferred embodiment in accordance with the above first to third
embodiment, the heavy chain variable domain comprises a region comprising an FR1,
a CDR1, an FR2, a CDR2 and an FR3 region and comprising the amino acid sequence of
SEQ ID NO: 1, and the light chain variable domain comprises a region comprising an
FR1, a CDR1, an FR2, a CDR2 and an FR3 region and comprising the amino acid sequence
of SEQ ID NO: 2.
[0128] Further, a monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof is provided, wherein the heavy chain variable domain comprises a CDR1
region comprising the amino acid sequence of SEQ ID NO: 3 and a CDR2 region comprising
the amino acid sequence of SEQ ID NO: 4, and wherein the light chain variable domain
comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 6 and a CDR2
region comprising the amino acid sequence of SEQ ID NO: 7. In a preferred aspect of
this embodiment, the antibody may have CDR3 sequences as defined in any of the embodiments
of the invention described above.
[0129] In another embodiment, the a monoclonal antibody capable of binding to human GDF-15,
or an antigen-binding portion thereof is provided, wherein the antibody or antigen-binding
portion thereof is capable of inhibiting cancer growth in a mammal, preferably a human
patient.
[0130] In another embodiment in accordance with the above embodiments, the invention relates
to an antigen-binding portion capable of binding to human GDF-15, wherein the antigen-binding
portion is a single-domain antibody (also referred to as "Nanobody™") . In one aspect
of this embodiment, the single-domain antibody comprises the CDR1, CDR2, and CDR3
amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.
In another aspect of this embodiment, the single-domain antibody comprises the CDR1,
CDR2, and CDR3 amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:
7, respectively. In a preferred aspect of this embodiment, the single-domain antibody
is a humanized antibody.
[0131] Preferably, the antibodies of the invention capable of binding to human GDF-15 or
the antigen-binding portions thereof have an equilibrium dissociation constant for
human GDF-15 that is equal to or less than 100 nM, less than 20 nM, preferably less
than 10 nM, more preferably less than 5 nM and most preferably between 0.1 nM and
2 nM.
[0132] In another embodiment of the invention, the antibody capable of binding to human
GDF-15 or the antigen-binding portion thereof binds to the same human GDF-15 epitope
as the antibody to human GDF-15 obtainable from the cell line B1-23 deposited with
the Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH (DMSZ) under the accession
No. DSM ACC3142. As described herein, antibody binding to human GDF-15 in accordance
with the present invention is assessed by surface plasmon resonance measurements as
a reference standard method, in accordance with the procedures described in Example
1. Binding to the same epitope on human GDF-15 can be assessed similarly by surface
plasmon resonance competitive binding experiments of the antibody to human GDF-15
obtainable from the cell line B1-23 and the antibody that is expected to bind to the
same human GDF-15 epitope as the antibody to human GDF-15 obtainable from the cell
line B1-23.
[0133] In a very preferred embodiment, the antibody is the monoclonal antibody capable of
binding to human GDF-15 obtainable from the cell line B1-23 deposited with the Deutsche
Sammlung fur Mikroorganismen und Zellkulturen GmbH (DMSZ) under the accession No.
DSM ACC3142 or an antigen-binding portion thereof.
[0134] In a preferred embodiment, the antibody capable of binding to human GDF-15 or the
antigen-binding portion thereof according to the invention is a humanized monoclonal
antibody or an antigen-binding portion thereof. For any given non-human antibody sequence
in accordance with the invention (i.e. a donor antibody sequence), humanized monoclonal
anti-human-GDF-15 antibodies of the invention or antigen-binding portions thereof
can be generated in accordance with techniques known in the art, as described above.
[0135] In a very preferred embodiment, the monoclonal antibody capable of binding to human
GDF-15 or antigen-binding portion thereof is a humanized antibody derived from the
monoclonal antibody capable of binding to human GDF-15 obtainable from the cell line
Bl-23 deposited with the Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH
(DMSZ) under the accession No. DSM ACC3142, or an antigen-binding portion thereof.
In a non-limiting aspect of this embodiment, the heavy chain variable domain of the
humanized antibody or antigen-binding portion thereof comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 5, and the light chain variable domain of the
humanized antibody or antigen-binding portion thereof comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 7. In a further non-limiting aspect of this
embodiment, the heavy chain variable domain of the humanized antibody or antigen-binding
portion thereof comprises or further comprises a CDR1 region comprising the amino
acid sequence of SEQ ID NO: 3 and a CDR2 region comprising the amino acid sequence
of SEQ ID NO: 4, and the light chain variable domain of the humanized antibody or
antigen-binding portion thereof comprises or further comprises a CDR1 region comprising
the amino acid sequence of SEQ ID NO: 6 and a CDR2 region comprising the amino acid
sequence of SEQ ID NO: 7.
[0136] Further, a monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof is provided, wherein the binding is binding to a conformational or
discontinuous epitope on human GDF-15 comprised by the amino acid sequences of SEQ
ID No: 25 and SEQ ID No: 26. In a preferred aspect of this embodiment, the antibody
or antigen-binding portion thereof is an antibody or antigen-binding portion thereof
as defined in any one of the above embodiments.
[0137] In another embodiment of the invention in accordance with the above embodiments,
the antibody capable of binding to human GDF-15 or the antigen-binding portion thereof
is a diabody. In one aspect of this embodiment, the diabody is bivalent and monospecific,
with two identical antigen binding sites for human GDF-15. In a second, alternative
aspect of this embodiment, the diabody is bivalent and bispecific, with one antigen
binding site being a binding site for human GDF-15, and the other antigen binding
site being a binding site for a different antigen. Non-limiting examples for the different
antigen according to this second aspect of this embodiment are i) cell surface antigens
that are co-expressed with GDF-15 at high levels on the same cancer (e.g. at higher
levels compared to a control sample of the same patient obtained from a non-cancerous
part of the tissue which is the tissue of origin of the cancer), and ii) cell surface
antigens on cells of the immune system which are known as useful antigens for the
recruitment of cells of the immune system to the tumor.
[0138] In still another embodiment of the invention in accordance with the above embodiments,
the antibody capable of binding to human GDF-15 or the antigen-binding portion thereof
is linked to a drug. In non-limiting aspects of this embodiment, the drug can be a
known anticancer agent and/or an immune-stimulatory molecule. Known anticancer agents
include alkylating agents such as cisplatin, carboplatin, oxaliplatin, mechlorethamine,
cyclophosphamide, chlorambucil, and ifosfamide; anti-metabolites such as azathioprine
and mercaptopurine; alkaloids such as vinca alkaloids (e.g. vincristine, vinblastine,
vinorelbine, and vindesine), taxanes (e.g. paclitaxel, docetaxel) etoposide and teniposide;
topoisomerase inhibitors such as camptothecins (e.g. irinotecan and topotecan); cytotoxic
antibiotics such as actinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicin,
idarubicin, epirubicin, bleomycin, plicamycin and mitomycin; and radioisotopes. Linking
of the antibodies or the antigen-binding portions thereof of the invention to anticancer
agents is expected to result in stronger cancer tumor growth inhibition compared to
the antibody without the anticancer agent, because the resulting conjugate will accumulate
at the site of the tumor due to the presence of GDF-15 in the tumor, leading to the
accumulation of the anticancer agent at the site of the tumor and to enhanced effects
of the anticancer agent on the tumor.
[0139] In a further embodiment in accordance with the above embodiments, the antibody capable
of binding to human GDF-15 or the antigen-binding portion thereof is modified by an
amino acid tag. Non-limiting examples of such tags include Polyhistidin (His-) tags,
FLAG-tag, Hemagglutinin (HA) tag, glycoprotein D (gD) tag, and c-myc tag. Tags may
be used for various purposes. For instance, they may be used to assist purification
of the antibody capable of binding to human GDF-15 or the antigen-binding portion
thereof, or they may be used for detection of the antibody or the antigen-binding
portion thereof (e.g. when used in diagnostic assays). Preferably, such tags are present
at the C-terminus or N-terminus of the antibody capable of binding to human GDF-15
or the antigen-binding portion thereof.
[0140] In a preferred embodiment of the present invention in accordance with the above embodiments,
the antibody capable of binding to human GDF-15 or the antigen-binding portion thereof
is capable of inhibiting cancer growth in a mammal, preferably a human patient.
[0141] In another preferred embodiment of the present invention in accordance with the above
embodiments, the human GDF-15 is recombinant human GDF-15 having the amino acid sequence
represented by SEQ ID No: 8.
[0142] In still another preferred embodiment of the present invention in accordance with
the above embodiments, the binding of the antibody capable of binding to human GDF-15
or the antigen-binding portion thereof is a binding to a conformational or discontinuous
epitope on human GDF-15.
[0143] Preferably, the monoclonal antibodies of the present invention capable of binding
to human GDF-15 or the antigen-binding portions thereof are isolated antibodies.
[0144] In a preferred embodiment of the above antibodies or antigen-binding portions thereof
according to the invention, the antibody has a size of more than 100 kDa, preferably
more than 110 kDa, more preferably more than 120 kDa, still more preferably more than
130 kDa, and most preferably more than 140 kDa. Preferably, the antibody is a full-length
antibody, more preferably a full-length IgG antibody.
[0145] The invention also relates to an expression vector comprising a nucleotide sequence
encoding the antibody or antigen-binding portion thereof as defined above.
[0146] Further, the present invention also provides a cell line capable of producing an
antibody or antigen-binding portion thereof according to the present invention.
[0147] In one embodiment, the cell line can be derived from any cell line that is known
in that art and suitable for the production of antibodies or antigen-binding portions
thereof.
[0148] In a preferred embodiment, the cell line is the cell line B1-23 deposited with the
Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (DMSZ) under the accession
No. DSM ACC3142.
[0149] In another preferred embodiment, the cell line contains an expression vector according
to the invention as defined above.
B) Pharmaceutical Compositions
[0150] In a further embodiment, the present invention relates to a pharmaceutical composition
comprising any of the antibodies or antigen-binding portions thereof as defined above.
[0151] Pharmaceutical compositions in accordance with the present invention are prepared
in accordance with known standards for the preparation of pharmaceutical compositions
containing antibodies and portions thereof.
[0152] For instance, the compositions are prepared in a way that they can be stored and
administered appropriately, e.g. by using pharmaceutically acceptable components such
as carriers, excipients or stabilizers.
[0153] Such pharmaceutically acceptable components are not toxic in the amounts used when
administering the pharmaceutical composition to a patient. The pharmaceutical acceptable
components added to the pharmaceutical compositions may depend on the particular intended
use of the pharmaceutical compositions and the route of administration.
C) Therapeutic Methods and Products for Use in these Methods
[0155] The present invention further relates to a method for treating cancer cachexia in
a mammal. The method comprises administering an antibody or antigen-binding portion
thereof as defined above, or a pharmaceutical composition as defined above to said
mammal. Alternatively, the present invention relates to an antibody or antigen-binding
portion thereof as defined above, or a pharmaceutical composition as defined above
for use in these methods. In a very preferred aspect of these embodiments, the mammal
is a human patient.
[0156] The present invention further relates to a method for treating cancer in a mammal.
The method comprises administering an antibody or antigen-binding portion thereof
as defined above, or a pharmaceutical composition as defined above to said mammal.
Alternatively, the present invention relates to an antibody or antigen-binding portion
thereof as defined above, or a pharmaceutical composition as defined above for use
in these methods. In a very preferred aspect of these embodiments, the mammal is a
human patient.
[0158] The present invention relates to several surprising advantages compared to the effects
observed in the art.
[0159] In particular, one main benefit of the invention lies in that the anti-GDF-15 antibodies
disclosed herein can be used to more effectively treat cancer-induced weight loss
and/or cancer cachexia.
[0160] For instance, the treatment with the antibodies according to the invention can completely
prevent cancer cachexia (when given prophylactically) or completely reverse cancer
cachexia (when given after the onset of cancer cachexia).
[0161] Moreover, the antibodies according to the invention can even increase the body weight
of the treated mammal during a prophylactic treatment for the prevention of cachexia.
Likewise, it is expected that in the course of a therapeutic treatment started after
the onset of cancer cachexia, the antibodies according to the invention can not only
reverse the loss in body weight, but also increase the body weight of the treated
mammal compared to its body weight before the onset of cancer cachexia.
[0162] This unexpected effect of the antibodies according to the invention may be beneficial
in various clinical situations. For instance, administration of many ingredients that
are pharmaceutically active against cancer (e.g. various chemotherapeutic drugs) can
lead to a loss of body weight of mammals including human patients. Such an additional
loss in body weight could be counteracted by the increase in body weight due to the
administration of the antibodies according to the invention. Therefore, the uses of
the antibodies according to the invention may be particularly advantageous and safe
for combination regimens with additional chemotherapeutic drugs. Similarly, the uses
of the antibodies according to the invention may be particularly advantageous for
mammals such as human patients that already had a low body weight prior to the onset
of cancer and/or prior to the onset of cancer cachexia. Patients with a low body weight
may for instance be cachectic patients, e.g. patients with a body-mass-index of less
than 18 kg/m
2.
[0163] Moreover, unexpectedly, according to the invention, the antibodies are not only effective
for the treatment of cancer cachexia, but also effective for the treatment of cancer.
[0164] Thus, the treatment methods and products for use of the antibodies according to the
invention are expected to be particularly beneficial for the treatment of cancer patient
sub-groups which suffer from cancer-induced weight loss and/or cancer cachexia, respectively.
[0165] However, the effects according to the invention are also expected to be advantageous
for the treatment of a complete patient group of a cancer referred to herein: By using
the antibodies according to the invention that are effective both against the cancer
itself and against cancer-induced weight loss and/or cancer cachexia, cancer and cancer
cachexia treatments may be simplified by using the same treatment for all cancer patients,
irrespective of whether or not they suffer from cancer-induced weight loss and/or
cancer cachexia. This is because due to the dual effects of the antibodies against
cancer and cancer cachexia, it is expected that these antibodies will obviate the
need for additional drugs for the treatment of cancer cachexia.
[0166] Likewise, due to the dual effects of the antibodies in accordance with the invention,
it may also become unnecessary to diagnose cancer-induced weight loss and/or cancer
cachexia. Hence it is expected that the overall costs of therapy and diagnosis will
be reduced.
[0167] Therefore, in a preferred embodiment of the above methods, or antibodies, antigen-binding
portions thereof or pharmaceutical compositions for use in these methods according
to the invention, the method for treating cancer cachexia is a method for completely
preventing or completely reverting cancer cachexia. In a more preferred embodiment
of this method, or the antibodies, antigen-binding portions thereof or pharmaceutical
compositions for use in this method, the method for treating cancer cachexia is a
method for completely preventing cancer cachexia. In an alternative more preferred
embodiment of this method, or the antibodies, antigen-binding portions thereof or
pharmaceutical compositions for use in this method, the method for treating cancer
cachexia is a method for completely reverting cancer cachexia.
[0168] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods according to the invention,
only mammals suffering from both
- i) the cancer, and
- ii) cancer cachexia
are treated in the method.
[0169] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods according to the invention,
the method increases body weight of the mammal compared to its body weight before
the onset of cancer cachexia. Preferably, the increase in body weight of the mammal
is at least 1.5%, preferably at least 2.5%, more preferably at least 5% compared to
its body weight before the onset of cancer cachexia.
[0170] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods according to the invention,
the method is a method for both treating cancer and treating cancer cachexia in the
same mammal.
[0171] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods according to the invention,
the antibody has a size of more than 100 kDa, preferably more than 110 kDa, more preferably
more than 120 kDa, still more preferably more than 130 kDa, and most preferably more
than 140 kDa. Preferably, the antibody is a full-length antibody, more preferably
a full-length IgG antibody.
[0172] In a further preferred embodiment of the above methods, or antibodies, antigen-binding
portions thereof or pharmaceutical compositions for use in these methods according
to the invention, the antibody has an Fc portion which is capable of binding to the
Fc receptor.
[0173] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods according to the invention,
the cancer cells of the mammal endogenously express GDF-15 and/or the cancer cells
of the mammal stimulate endogenous expression of GDF-15 in non-cancerous cells of
the mammal.
[0174] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods according to the invention,
the cancer cells of the mammal are characterized in that they endogenously express
GDF-15.
[0175] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods according to the invention,
the mammal is human patient.
[0176] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods according to the invention,
the human GDF-15 is recombinant human GDF-15 having the amino acid sequence represented
by SEQ ID No: 8.
[0177] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods, the human patient
has elevated GDF-15 levels in blood serum before administration. In a patient sub-group
having elevated GDF-15 levels in blood serum, the treatment methods according to the
invention are expected to be particularly effective at inhibiting cancer growth. In
the most preferred aspect of this embodiment, GDF-15 levels are GDF-15 protein levels
measured using the antibody according to the invention obtainable from the hybridoma
cell line B1-23 deposited with the Deutsche Sammlung für Mikroorganismen und Zellkulturen
GmbH (DSMZ) under the accession No. DSM ACC3142, preferably measured by immunochemistry.
[0178] In another embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods, the antibody or antigen-binding
portion thereof is the sole ingredient pharmaceutically active against cancer used
in the method.
[0179] In an alternative embodiment of the above methods, or antibodies, antigen-binding
portions thereof or pharmaceutical compositions for use in these methods, the antibody
or antigen-binding portion thereof is used in combination with one or more further
ingredients pharmaceutically active against cancer. In one aspect of this embodiment,
the one or more further ingredients pharmaceutically active against cancer is a known
anticancer agent and/or an immune-stimulatory molecule as defined above. Thus, the
anticancer agent can for instance be selected from alkylating agents such as cisplatin,
carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide;
anti-metabolites such as azathioprine and mercaptopurine; alkaloids such as vinca
alkaloids (e.g. vincristine, vinblastine, vinorelbine, and vindesine), taxanes (e.g.
paclitaxel, docetaxel) etoposide and teniposide; topoisomerase inhibitors such as
camptothecins (e.g. irinotecan and topotecan); cytotoxic antibiotics such as actinomycin,
anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin,
plicamycin and mitomycin; and radioisotopes. Due to the increasing effect of the antibodies
according to the invention on body weight of the mammals including human patients,
these combined uses of the antibodies or antigen-binding portions thereof and the
ingredients pharmaceutically active against cancer are expected to be particularly
safe, because they may compensate a possible additional weight loss resulting from
the administration of the ingredients pharmaceutically active against cancer.
[0180] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods, the cancer is selected
from the group consisting of brain cancers including glioma, cancers of the nervous
system, melanoma, lung cancer, lip and oral cavity cancer, hepatic carcinoma, leukemia,
Hodgkin lymphoma, Non-Hodgkin lymphoma, bladder cancer, cervix uteri cancer, corpus
uteri cancer, testis cancer, thyroid cancer, kidney cancer, gallbladder cancer, multiple
myeloma, nasopharynx cancer, larynx cancer, pharynx cancer, oesophagus cancer, gastrointestinal
tumors including stomach and colorectal cancer, pancreatic cancer, prostate cancer,
ovarian cancer and breast cancer, preferably from the group consisting of melanoma,
prostate cancer, breast cancer, brain cancers including glioma, colorectal cancer,
stomach cancer, oesophagus cancer and ovarian cancer, and most preferably is melanoma.
In one embodiment the cancer is selected from the above group, which further comprises
endometrial cancer, such as endometrial carcinoma, breast cancer including subtypes
of breast cancer, in particular triple-negative breast cancer and bladder cancer such
as urothelial cell carcinoma.
[0181] In another preferred embodiment of the above methods, or antibodies, antigen-binding
portions thereof or pharmaceutical compositions for use in these methods, the tumor
or tumors formed by the cancer have higher human GDF-15 levels prior to administration
compared to a control sample of the same patient obtained from a non-cancerous part
of the tissue which is the tissue of origin of the cancer, preferably 1.2-fold higher
levels, more preferably 1.5-fold higher levels, still more preferably 2-fold higher
levels and most preferably 5-fold higher levels. In a patient sub-group having higher
GDF-15 levels in the tumor or tumors formed by the cancer compared to the above control
sample, the treatment methods according to the invention are expected to be particularly
effective at inhibiting cancer growth.
[0182] In a very preferred embodiment of the above methods, or antibodies, antigen-binding
portions thereof or pharmaceutical compositions for use in these methods, the method
for treating cancer comprises inhibiting cancer growth. In a preferred aspect of this
embodiment, cancer growth is stopped. In a more preferred aspect, the cancer shrinks.
[0183] In a preferred embodiment of the above methods, or antibodies, antigen-binding portions
thereof or pharmaceutical compositions for use in these methods, the method for treating
cancer comprises the induction of killing of cancer cells by NK cells and CD8+ T cells
in the human patient. Due to their capability of preventing GDF-15 mediated down-regulation
of the known immune surveillance regulator NKG2D, the antibodies or antigen-binding
portions thereof according to the invention are expected to restore immune surveillance
and induce the killing of cancer cells by NK cells and CD8+ T cells, in addition to
effects of the antibodies or antigen-binding portions thereof that are independent
of the immune system.
D) Kits
[0184] The present invention also provides kits comprising the pharmaceutical compositions
as defined above.
[0185] In one embodiment, the kits are kits for use in the methods according to the invention
as defined above.
[0186] In further embodiments, the present invention also provides a diagnostic kit comprising
any of the antibodies or antigen-binding portions thereof according to the invention.
[0187] In one embodiment, the diagnostic kit may be used to detect whether the tumor or
tumors of a cancer patient formed by the cancer have higher human GDF-15 levels compared
to a control sample of the same patient obtained from a non-cancerous part of the
tissue which is the tissue of origin of the cancer.
[0188] In another embodiment, the diagnostic kit may be used to detect whether a human cancer
patient has elevated GDF-15 levels in blood serum.
E) Sequences
[0189] The amino acid sequences referred to in the present application are as follows (in
an N-terminal to C-terminal order; represented in the one-letter amino acid code):
SEQ ID No: 3 (Heavy Chain CDR1 Region Peptide Sequence of monoclonal anti-human GDF-15
mAb-Bl-23):
GFSLSTSGMG
SEQ ID No: 4 (Heavy Chain CDR2 Region Peptide Sequence of monoclonal anti-human GDF-15
mAb-Bl-23):
IYWDDDK
SEQ ID No: 5 (Heavy Chain CDR3 Region Peptide Sequence of monoclonal anti-human GDF-15
mAb-B1-23):
ARSSYGAMDY
SEQ ID No: 6 (Light Chain CDR1 Region Peptide Sequence of monoclonal anti-human GDF-15
mAb-B1-23):
QNVGTN
Light Chain CDR2 Region Peptide Sequence of monoclonal anti-human GDF-15 mAb-B1-23:
SAS
SEQ ID No: 7 (Light Chain CDR3 Region Peptide Sequence of monoclonal anti-human GDF-15
mAb-B1-23):
QQYNNFPYT




SEQ ID No: 11 (Flag peptide): DYKDDDDKGG
SEQ ID No: 12 (HA peptide): YPYDVPDYAG
SEQ ID No: 13 (peptide derived from human GDF-15):
ELHLRPQAARGRR
SEQ ID No: 14 (peptide derived from human GDF-15):
LHLRPQAARGRRR
SEQ ID No: 15 (peptide derived from human GDF-15):
HLRPQAARGRRRA
SEQ ID No: 16 (peptide derived from human GDF-15):
LRPQAARGRRRAR
SEQ ID No: 17 (peptide derived from human GDF-15):
RPQAARGRRRARA
SEQ ID No: 18 (peptide derived from human GDF-15):
PQAARGRRRARAR
SEQ ID No: 19 (peptide derived from human GDF-15):
QAARGRRRARARN
SEQ ID No: 20 (peptide derived from human GDF-15):
MHAQIKTSLHRLK
SEQ ID No: 25 (GDF-15 peptide comprising part of the GDF-15 Epitope that binds to
B1-23):
EVQVTMCIGACPSQFR
SEQ ID No: 26 (GDF-15 peptide comprising part of the GDF-15 Epitope that binds to
B1-23):
TDTGVSLQTYDDLLAKDCHCI
F) Examples
[0191] The present invention is illustrated by the following nonlimiting Examples:
Example 1: Generation and characterization of the murine GDF-15 Antibody B1-23, and generation
of chimeric and humanized antibodies.
[0192] The antibody Bl-23 was generated in a GDF-15 knock out mouse. Recombinant human GDF-15
(SEQ ID No: 8) was used as the immunogen.
[0193] The hybridoma cell line B1-23 producing mAb-B1-23 was deposited with the Deutsche
Sammlung für Mikroorganismen und Zellkulturen GmbH (DMSZ) under the accession No.
DSM ACC3142, in accordance with the Budapest Treaty.
[0194] By means of a commercially available test strip system, B1-23 was identified as an
IgG2a (kappa chain) isotype. Using surface plasmon resonance measurements, the dissociation
constant (Kd) was determined as follows:
Binding of the monoclonal anti-human-GDF-15 antibody anti-human GDF-15 mAb-B1-23 was
measured by employing surface plasmon resonance measurements using a Bio-Rad® ProteOn™
XPR36 system and Bio-Rad® GLC sensor chips:
For preparing the biosensors recombinant mature human GDF-15 protein was immobilized
on flow cells 1 and 2. On one flow cell recombinant GDF-15 derived from Baculvirus-transfected
insect cells (HighFive insect cells) and on the other recombinant protein derived
from expression in E. coli was used. The GLC sensor chip was activated using Sulfo-NHS
(N-Hydroxysulfosuccinimide) and EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride) (Bio-Rad® ProteOn™ Amine Coupling Kit) according to the manufacturer's
recommendation, the sensor surface was subsequently loaded with the proteins up to
a density of about 600RU (1Ru = 1pg mm
-2). The non-reacted coupling groups were then quenched by perfusion with 1M ethanolamine
pH 8.5 and the biosensor was equilibrated by perfusing the chip with running buffer
(10M HEPES, 150mM NaCl, 3.4mM EDTA, 0.005% Tween®20, pH 7.4, referred to as HBS150).
As controls two flow cells were used, one empty with no protein coupled and one coupled
with an non-physiological protein partner (human Interleukin-5), which was immobilized
using the same coupling chemistry and the same coupling density. For interaction measurements
anti-human GDF-15 mAb-B1-23 was dissolved in HBS150 and used in six different concentrations
as analyte (concentration: 0.4, 0.8, 3, 12, 49 und 98 nM). The analyte was perfused
over the biosensor using the one-shot kinetics setup to avoid intermittent regeneration,
all measurements were performed at 25°C and using a flow rate of 100µl min
-1. For processing the bulk face effect and unspecific binding to the sensor matrix
was removed by subtracting the SPR data of the empty flow cell (flow cell 3) from
all other SPR data. The resulting sensogram was analyzed using the software ProteOn
Manager version 3.0. For analysis of the binding kinetics a 1:1 Langmuir-type interaction
was assumed. For the association rate constant a value of 5.4±0.06x10
5 M
-1s
-1 (k
on) and for the dissociation rate constant a value of 4.3±0.03x10
-4 s
-1 (k
off) could be determined (values are for the interaction of anti-human GDF-15 mAb-B1-23
with GDF-15 derived from insect cell expression). The equilibrium dissociation constant
was calculated using the equation K
D = k
off/k
on to yield a value of about 790pM. Affinity values for the interaction of GDF-15 derived
from E. coli expression and the anti-human GDF-15 mAb-B1-23 differ by less than a
factor of 2, rate constants for GDF-15 derived from insect cells and E. coli deviate
by about 45% and are thus within the accuracy of SPR measurements and likely do not
reflect a real difference in affinity. Under the conditions used the anti-human GDF-15
mAb-B1-23 shows no binding to human interleukin-5 and thus confirms the specificity
of the interaction data and the anti-human GDF-15 mAb-B1-23.
[0195] The amino acid sequence of recombinant human GDF-15 (as expressed in Baculovirus-transfected
insect cells) is:

[0196] Thus, using surface plasmon resonance measurements, the dissociation constant (Kd)
of 790pM was determined. As a comparison: the therapeutically used antibody Rituximab
has a significantly lower affinity (Kd = 8 nM).
[0197] From the murine anti-human GDF-15 mAb-B1-23, a chimeric anti-human GDF-15 mAb-B1-23
antibody according to the invention was generated by replacing constant domains of
the murine antibody with the constant domains of a human IgG1 antibody (trastuzumab
backbone). The amino acid sequence of the heavy chain of this chimeric antibody is
shown in SEQ ID No: 33, and the amino acid sequence of the light chain of this chimeric
antibody is shown in SEQ ID No: 36.
[0198] From the chimeric anti-human GDF-15 mAb-B1-23, a humanized anti-human GDF-15 mAb-B1-23
antibody according to the invention was developed by humanizing the variable domains
of the chimeric antibody, i.e. by replacing the framework regions of the chimeric
antibody with human sequences. The amino acid sequence of the heavy chain of this
humanized antibody is shown in SEQ ID No: 27, and the amino acid sequence of the light
chain of this humanized antibody is shown in SEQ ID No: 30. This antibody is referred
to as H1L5 anti-GDF-15 antibody or humanized B1-23-H1L5 antibody or H1L5 antibody.
[0199] In order to generate the above-mentioned chimeric anti-human GDF-15 mAb-B1-23 antibody
and the humanized B1-23-H1L5 antibody as indicated above, the cDNAs encoding the antibody
sequences were optimized, and the genes were synthesized. The gene sequences were
then cloned into a cloning/expression vector system. From these vectors, plasmid DNA
with low endotoxin levels was synthesized.
[0200] The plasmid DNA was then transiently transfected into CHO cells, followed by an analysis
and quantification of antibody expression using a protein A biosensor. The cDNA of
candidate cultures for antibody expression was sequenced. The obtained monoclonal
antibodies were analyzed (see Examples 7 to 9).
Example 2: Antagonization of GDF-15 Mediated Effects with mAB B1-23
[0201]
- a) The NKG2D (Natural Killer Group 2D) receptor, which is expressed on NK cells and
CD8+ T cells, is known to play an important role in the immune surveillance against
tumors. Transformed as well as viral infected cells express ligands, which bind to
the NKG2D receptor, thereby activating the cytotoxic effector functions of the described
immune cells. In that way transformed cells can be detected and eliminated by the
immune system. After treatment of immune cells with either recombinant human GDF-15
or tumor cell secreted GDF-15 in vitro for 72 hours, the expression level of NKG2D
on the cell surface of lymphocytes was downregulated (Figure 1) . After 72 hours incubation
the immune cells were stained with the following FACS-antibodies: anti CD3, anti CD56,
anti-NKG2D. Using this antibody combination, the experiment focused on NK cells and
their NKG2D surface expression. The low NKG2D level on immune cells led to an impaired
tumor/target cell lysis. The GDF-15 mediated downregulation of NKG2D was prevented
by mAb B1-23.
It is therefore concluded that human GDF-15 downregulates expression of NKG2D on the
cell surface of lymphocytes and thereby downregulates immune surveillance against
tumors. By binding to human GDF-15, the antibodies of the present invention are capable
of preventing GDF-15 mediated downregulation of NKG2D and should be capable of restoring
immune surveillance and inducing the killing of cancer cells by NK cells and CD8+
T cells. Given that the CDR regions of the mAb B1-23 antibody correspond to CDR regions
of the chimeric and humanized antibodies, it is expected that the functional properties
including the binding properties of these antibodies are similar.
- b) The treatment of the ovarian cancer cell line SK-OV-3 with recombinant GDF-15 led
to the phosphorylation of AKT. AKT is a molecule, which is part of the PI3K-pathway
and contributes to the activation and proliferation of cells. In this experiment SK-OV-3
cells were treated with 10 ng/ml recombinant GDF-15 for 10 min at 37°C, 5% CO2. 5
minutes preincubation of 2 µg mAb-B1-23 with 10 ng/ml GDF-15 at 37°C blocked the GDF-15 mediated AKT-phosphorylation
(Figure 2). This showed the neutralizing effect of mAb-B1-23.
- c) Treatment of immune cells with recombinant GDF-15 led to the phosphorylation of
JNK, a kinase, which is activated either by cytokines or by stress. Antagonization
of 10 ng/ml GDF-15 with 2 µg mAb-B1-23 (5 minute preincubation at 37°) blocked the GDF-15 mediated JNK1/2-phosphorylation
(Figure 3) .
Example 3: Inhibition of Cancer Cell Proliferation Using mAb B1-23
[0202] Data generated with B1-23 showed an antiproliferative effect of the antibody on cancer
cells in vitro. The strongest antiproliferative effect was observed using the prostate
cancer cell line LnCap, which produces lots of GDF-15. A metabolic assay (Alamar Blue®
assay) showed a decrease of proliferation of 30% after 72hrs when mAb-B1-23 was present,
compared with the control group, where the antibody was not applied. Since cytotoxic
effects of the antibody have been excluded in different assays, this effect proves
a significantly decreased cell division rate after blockade of GDF-15.
Example 4: mAb B1-23 inhibits Growth of tumors in vivo
[0203] The following in vivo study was carried out:
To assess an anti-tumor effect of B1-23 in vivo, Balb/c
nu/nu nude mice were used in a xenograft setting with the melanoma cell line UACC-257.
The mice were treated either with the antibody B1-23 or with PBS. Each treatment cohort
contained 10 Balb/c
nu/nu nude mice.
[0204] Prior to injection, the UACC-257 melanoma cells were grown in complete medium, excluding
any contamination. The cells were harvested when 70-80% confluence was reached in
the cell culture flask. Cells were then washed with PBS and counted. 1x10
7 viable cells were suspended in PBS.
[0205] The first injection/treatment was administered in 6 week old Balb/c
nu/nu nude mice. The inoculation area of the mice was cleaned with ethanol. The UACC 257
cells were mixed and drawn into a syringe without a needle, in order to avoid negative
pressure on the tumor cells. The cell suspension containing 1x10
7 cells in PBS was injected subcutaneously (s.c.) into the lower flank of the mice.
[0206] The intraperitoneal (i.p.) injection of either B1-23 (25mg/kg body weight) or the
same volume of PBS started immediately after the tumor cell inoculation (defined as
day 1) and was administered twice a week. The tumors were grown for 48 days. The tumor
diameters were measured with a caliper and the tumor volume in mm
3 was calculated by the formula:

[0207] The results which were obtained from the study are shown in Figure 4.
[0208] As demonstrated in the Figure, the tumor size of the animal cohort treated with B1-23
was significantly decreased, compared to the PBS control group.
[0209] Given that the CDR regions of the mAb Bl-23 antibody correspond to CDR regions of
the chimeric and humanized antibodies, it is expected that the functional properties
including anti-cancer effects of these antibodies are similar.
Example 5: mAb B1-23 recognizes a conformational or a discontinuous epitope of human GDF-15
[0210] Epitope Mapping: Monoclonal mouse antibody GDF-15 against 13mer linear peptides derived
from GDF-15
Antigen: GDF-15:
[0211] GSGSGSGMPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFR ELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLP
EASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSA RPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQV
TMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYD DLLAKDCHCI
GSGSGSG (322 amino acids with linker) (SEQ ID No: 10)
[0212] The protein sequence was translated into 13mer peptides with a shift of one amino
acid. The C- and N-termini were elongated by a neutral GSGS linker to avoid truncated
peptides (bold letters).
Control Peptides:
[0213] Flag: DYKDDDDKGG (SEQ ID No:13), 78 spots; HA: YPYDVPDYAG (SEQ ID No:14), 78 spots
(each array copy)
Peptide Chip Identifier:
[0214] 000264_01 (10/90, Ala2Asp linker)
Staining Conditions:
[0215] Standard buffer: PBS, pH 7.4 + 0.05% Tween®20
Blocking buffer: Rockland blocking buffer MB-070
Incubation buffer: Standard buffer with 10% Rockland blocking buffer MB-070
Primary sample: Monoclonal mouse antibody GDF-15 (1 µg/µl) : Staining in incubation
buffer for 16 h at 4°C at a dilution of 1:100 and slight shaking at 500 rpm
Secondary antibody: Goat anti-mouse IgG (H+L) IRDye680, staining in incubation buffer
with a dilution of 1:5000 for 30 min at room temperature (RT)
Control antibodies: Monoclonal anti-HA (12CA5)-LL-Atto 680 (1:1000), monoclonal anti-FLAG(M2)-FluoProbes752
(1:1000); staining in incubation buffer for 1 h at RT
Scanner:
[0216] Odyssey® Imaging System, LI-COR Biosciences
Settings: offset: 1mm; resolution: 21 µm; intensity green/red: 7/7
Results:
[0217] After 30 min pre-swelling in standard buffer and 30 min in blocking buffer, the peptide
array with 10, 12 and 15mer B7H3-derived linear peptides was incubated with secondary
goat anti-mouse IgG (H+L) IRDye680 antibody only at a dilution of 1:5000 for 1h at
room temperature to analyze background interactions of the secondary antibody. The
PEPperCHIP® was washed 2x1 min with standard buffer, rinsed with dist. water and dried
in a stream of air. Read-out was done with Odyssey® Imaging System at a resolution
of 21 µm and green/red intensities of 7/7: We observed a weak interaction of arginine-rich
peptides (ELHLRPQAARGRR (SEQ ID No:15), LHLRPQAARGRRR (SEQ ID No:16), HLRPQAARGRRRA
(SEQ ID No:17), LRPQAARGRRRAR (SEQ ID No:18), RPQAARGRRRARA (SEQ ID No:19), PQAARGRRRARAR
(SEQ ID No:20) and QAARGRRRARARN (SEQ ID No:21)) that are known as frequent binders,
and with the basic peptide MHAQIKTSLHRLK (SEQ ID No:22) due to ionic interactions
with the charged antibody dye.
[0218] After pre-swelling for 10 min in standard buffer, the peptide microarray was incubated
overnight at 4 °C with monoclonal mouse antibody GDF-15 at a dilution of 1:100. Repeated
washing in standard buffer (2x1 min) was followed by incubation for 30 min with the
secondary antibody at a dilution of 1:5000 at room temperature. After 2x10 sec. washing
in standard buffer and short rinsing with dist. water, the PEPperCHIP® was dried in
a stream of air. Read-out was done with Odyssey® Imaging System at a resolution of
21 µm and green/red intensities of 7/7 before and after staining of control peptides
by anti-HA and anti-FLAG(M2) antibodies.
[0219] It was shown that none of the linear 13mer peptides derived from GDF-15 interacted
with monoclonal mouse antibody GDF-15 even at overregulated intensities. Staining
of Flag and HA control peptides that frame the array, however, gave rise to good and
homogeneous spot intensities.
Summary:
[0220] The Epitope Mapping of monoclonal mouse GDF-15 antibody against GDF-15 did not reveal
any linear epitope with the 13mer peptides derived from the antigen. According to
this finding it is very likely that monoclonal mouse antibody GDF-15 recognizes a
conformational or a discontinuous epitope with low affinity of partial epitopes. Due
to the obvious absence of any GDF-15 signal above the background staining of the secondary
antibody only, quantification of spot intensities with PepSlide® Analyzer and subsequent
peptide annotation were omitted.
Example 6: Structural identification of peptide ligand epitopes by mass spectrometric epitope
excision and epitope extraction
[0222] For preparation of the antibody column, the antibody Bl-23 was added to NHS-activated
6-aminohexanoic acid coupled sepharose. The sepharose-coupled antibody B1-23 was then
loaded into a 0,8 ml microcolumn and washed with blocking and washing buffers.
Epitope extraction experiment:
[0223] Recombinant human GDF-15 was digested with trypsin for 2h at 37°C (in solution),
resulting in different peptides, according to the trypsin cleavage sites in the protein.
After complete digestion, the peptides were loaded on the affinity column containing
the immobilized antibody B1-23. Unbound as well as potentially bound peptides of GDF-15
were used for mass spectrometry analysis. An identification of peptides by means of
mass spectrometry was not possible. This was a further indicator that the binding
region of GDF-15 in the immune complex B1-23 comprises a discontinuous or conformational
epitope. In case of a continuous linear epitope, the digested peptides should bind
its interaction partner, unless there was a trypsin cleavage site in the epitope peptide.
A discontinuous or conformational epitope could be confirmed by the epitope excision
method described in the following part.
Epitope excision experiment:
[0224] The immobilized antibody B1-23 on the affinity column was then incubated with recombinant
GDF-15 for 2h. The formed immune complex on the affinity column was then incubated
with trypsin for 2h at 37°C. The cleavage resulted in different peptides derived from
the recombinant GDF-15. The immobilized antibody itself is proteolytically stable.
The resulting peptides of the digested GDF-15 protein, which were shielded by the
antibody and thus protected from proteolytic cleavage, were eluted under acidic conditions
(TFA, pH2), collected and identified by mass spectrometry.
[0225] The epitope excision method using MS/MS identification resulted in the following
peptides:
Peptide |
Position in sequence |
Mass |
Ion/Charge |
EVQVTMCIGACPSQFR (SEQ ID No: 25) |
40-55 |
1769.91 |
590.50(3+) |
TDTGVSLQTYDDLLAKDCHCI (SEQ ID No: 26) |
94-114 |
2310,96 |
771:33(3+) |
[0226] The part of human GDF-15, which binds the antibody B1-23, comprises a discontinuous
or conformational epitope. Mass spectrometry identified 2 peptides in the GDF-15 protein,
which are responsible for the formation of the immune complex. These peptides are
restricted to the positions 40-55 (EVQVTMCIGACPSQFR) and 94-114 (TDTGVSLQTYDDLLAKDCHCI)
in the GDF-15 amino acid sequence. Thus, these two peptides comprise an epitope of
the GDF-15 protein that binds to the antibody B1-23.
[0227] Again, since the CDR regions of the mAb Bl-23 antibody correspond to CDR regions
of the chimeric and humanized antibodies, it is expected that the binding properties
of these antibodies are similar.
Example 7: Treatment of cancer-induced weight loss with anti-GDF-15 antibodies. In the underlying
animal study No. 140123, 10 Balb/c nu/nu mice per treatment group were subcutaneously
inoculated with 10x106 UACC-257 cells per animal in a 1:1 volume ratio with matrigel (100 µl cells + 100 µl matrigel). The animals were treated on the same day with the respective antibodies,
as indicated below:
[0228]
Study groups 1-6 (10 animals per group) |
Amounts of substances (for 45 days) |
1. Dacarbazine* (reference, Lot. No.: C120522C) |
80 mg |
2. PBS (SIGMA, Lot. No.: RNBD0341) |
30 ml |
3. B1-23 anti-GDF-15 antibody (murine, Lot. No.: 515980) |
75 mg |
4. Chimeric B1-23 anti-GDF-15 antibody (chimeric; Lot.:PR0057) |
75 mg |
5. H1L5 anti-GDF-15 antibody |
|
(humanized B1-23, Lot.:PR3176) |
75 mg |
6. B12 Isotype control antibody (Isotype antibody, Lot. No.: ID3195) |
75 mg |
*Detidemac 500 mg (exp.: 03/2015) |
[0229] The dacarbazine group (group 1) served as a reference group /positive control for
tumor growth arrest (cytostatic drug for the treatment of malignant melanoma in humans).
[0230] The PBS group (group 2) served as a growth control/vehicle control group, because
all used substances of the other groups were administered in PBS.
[0231] The group of the murine Bl-23 lead candidate antibody (group 3) served as reference
group for a comparison with the chimeric B1-23 antibody and with the humanized B1-23
H1L5 (groups 4 and 5).
[0232] Group 4 is the group of the chimerized B1-23 lead candidate antibody, which contains
murine variable domains and constant domains of a human IgG1 antibody (trastuzumab
backbone).
[0233] Group 5 is the group of the H1L5 humanized B1-23 lead candidate antibody, which contains
humanized frameworks within the murine variable regions and constant domains of a
human IgG1 antibody (trastuzumab backbone).
[0234] Group 6 is the group of the B12 isotype antibody. For this isotype control group,
the antibody B12 (Lot. No.: ID3195) was produced by the company Evitria AG. B12 binds
to an HIV antigen and should therefore neither bind to antigens in nude mice nor to
antigens of the human tumor. B12 was selected as a highly suitable isotype control,
because the immunoglobulin backbone of B12 also consists of the human IgG1 antibody
trastuzumab and is therefore almost identical to the chimeric B1-23 and the H1L5 humanized
B1-23 antibodies, except for their variable regions.
[0235] The study was carried out in a double-blinded manner for the treatment with the antibodies
and for the treatment with PBS.
[0236] In groups 1, 2 and 6 which did not receive anti-GDF-15 antibodies, more than 10%
body weight loss was observed (i.e. weight loss to a relative body weight of less
than 90% compared to day 0). In contrast, in the groups which had received treatment
with the anti-GDF-15 antibodies B1-23, chimeric B1-23 and humanized B1-23-H1L5, respectively,
an increase in body weight was observed (Figure 5).
Thus, surprisingly, treatment with all of the tested anti-GDF-15 antibodies completely
prevents cancer-induced weight loss in mice. This effect was significant for all of
the groups treated with anti-GDF-15 antibodies (two-way ANOVA; p<0.05).
[0237] It is also noteworthy that the mice of the groups that did not receive treatment
with anti-GDF-15 antibodies exhibited a weight loss of more than 10%. In humans, a
weight loss of as little as 5% over a period of 6 months is considered as being indicative
of cancer cachexia (
Fearon K. et al.: Definition and classification of cancer cachexia: an international
consensus. Lancet Oncol. 2011 May; 12(5):489-95.). Given the larger weight loss of the mice observed in the present study which even
exceeded 10%, it is expected that the mice in the study, which did not receive treatment
with anti-GDF-15 antibodies, not only exhibited weight loss but also exhibited cancer
cachexia. This effect is completely prevented by the anti-GDF-15 antibodies tested.
It is therefore expected that the anti-GDF-15 antibodies in accordance with the invention
are capable of both treating cancer-induced weight loss and treating cancer cachexia.
[0238] Notably, the extent of weight loss did not correlate with the respective tumor size
(r
2=10
-6). If the prevention of weight loss were only a secondary effect resulting from the
inhibition of cancer growth and the smaller tumor sizes, a correlation between tumor
size and weight loss would be expected. Thus, the lack of such correlation shows that
uses of the anti-GDF-15 antibodies according to the invention result in two independent
treatment effects:
- an inhibition of cancer growth, and
- a prevention of weight loss as an additional effect, which is independent from the
inhibition of cancer growth, and which is expected to reflect a prevention of cancer
cachexia.
Despite their mechanistic independence, it was observed that these effects can occur
simultaneously in the same animals.
[0239] In addition to evaluating the mean body weight of the mice, the feed consumption
of the mice was evaluated by pairwise comparisons of the study groups (Table 1). Notably,
the feed consumption of the mice in the anti-GDF-15 antibody groups (B1-23, chimeric
B1-23 and humanized B1-23-H1L5) was significantly higher than the feed consumption
of the mice in the groups which did not receive the anti-GDF-15 antibodies.
Table 1:
|
Feed consumption per mouse and day |
vs. chimeric B1-23 |
vs. humanized. Bl-23 |
vs. B1-23 |
dacarbazine |
2.8 ± 0.2 g |
** |
** |
** |
PBS |
2.6 ± 0.4 g |
** |
** |
** |
Chimeric B1-23 |
3.5 ± 0.2 g |
-- |
n.s. |
n.s. |
B12 |
2.7 ± 0.2 g |
*** |
*** |
*** |
humanized Bl-23 |
3.4 ± 0.2 g |
n.s. |
-- |
n.s. |
Bl-23 |
3.6 ± 0.2 g |
n.s. |
n.s. |
- |
(*p<0.05; **p<0.01; ***p<0.001 as assessed by unpaired two-sided Student's t-test) |
[0240] Table 1: Comparative evaluation of the feed consumption between the different treatment
groups. For the measured time intervals (day 17-20, day 20-24, day 24-27, day 27-31,
day 31-34), the average feed consumption per mouse and day was calculated for each
respective group. The values are indicated together with their standard deviation.
[0241] The quality of the humanized anti-GDF-15 antibody B1-23-H1L5 used in the study was
tested by using gel electrophoresis and coomassie staining of the antibodies (see
Figure 6). Notably, the band of the humanized anti-GDF-15 antibody B1-23-H1L5 was
sharp and clear, whereas the bands of the murine B1-23 anti-GDF-15 antibody and the
B12 control antibody appeared less sharp and at a higher molecular weight. This suggests
that the humanized anti-GDF-15 antibody B1-23-H1L5 is not prone to aggregation, and
that some aggregation may have shifted the molecular weight of the other antibodies
to higher values.
[0242] Additionally, by using a colorimetric assay, it was confirmed that all anti-GDF-15
antibodies used in the study bound to GDF-15 in a concentration-dependent manner.
To determine the binding of Bl-23 antibody variants to GDF-15, a colorimetric ELISA
experiment was performed. The B12 antibody served as an isotype antibody, which does
not bind to human GDF-15. Therefore, Maxisorp 96 well plates (Nunc) were coated with
hrGDF-15 (25 ng protein per well, 50
µl volume) over night at 4°C. The following day, plates were washed to remove unbound
protein (3 times with 150
µl of PBS 0.05% Tween®) and nonspecific binding sites were blocked with 150
µl of PBS 1% BSA for 2 hours at room temperature. Again, plates were washed and different
variants of B1-23 test antibodies were applied (50
µl volume). To inquire specificity of the antibody binding, endpoint dilution was performed
starting from 333 ng/ml and 1:3 serial dilution. As background control, PBS 1% BSA
was applied. Following binding for 1 hour at room temperature, wells were washed as
described above. As secondary antibody HRP conjugated Anti-human IgG (Life technologies,
1:5000) was applied for 1 hour at room temperature. Wells were washed as described
above to remove unbound secondary antibody. For detection, 50
µl of peroxide substrate (TMB 1:100 in 0.1 M sodium acetate pH6) were added and following
10 minutes of incubation, 50
µl of stop solution (2N H
2SO
4) were added. As negative controls, wells without GDF-15 coating and wells without
secondary antibody were included. For analysis, optical densitiy at 450 nm was quantified
using the ELISA reader (Tecan Sunrise) and the corresponding Magellan software. It
was observed that in comparison to the B12 antibody, the humanized H1L5 antibody,
the chimeric B1-23 antibody and the murine B1-23 antibody exhibited a clearly concentration-dependent
binding to GDF-15.
Example 8: Determination of Kd values of anti-GDF-15 antibodies. The Kd values of different
anti-GDF-15 antibodies were compared using Surface Acoustic Wave (SAW) gold chip biosensors
technology (SAW Instruments GmbH, Schwertberger Str. 16, D-53177 Bonn, Germany):
[0243]
Antibody: |
Kd value (nM) |
B1-23 anti-GDF-15 antibody (murine, IgG2a) |
28.8 nM |
Chimeric B1-23 anti-GDF-15 antibody (chimerized, human IgG1) |
14 nM |
H1L5 humanized B1-23 (humanized, human IgG1) |
5,62 nM |
Rituxumab (control antibody) |
1116 nM |
Herceptin (control antibody) |
No binding |
[0244] The murine antibody (B1-23) as well as the chimeric B1-23 antibody were present in
purified form. The H1L5 humanized Bl-23 antibody was a serum-free CHO cell culture
supernatant. The Kd value of the murine B1-23 deviates from the Kd values determined
by Biacore® analyses (surface plasmon resonance) by a factor of 35.
[0245] This deviation may - apart from the differences in the measurement methods - be explained
by a reduced availability of free murine B1-23 antibody, since it was found that this
antibody can form aggregates in its form as mouse antibody. The solution of the murine
B1-23 antibody was therefore stabilized by addition of 0.2% BSA. Therefore, binding
of the antibody to albumin may have reduced the availability of the murine B1-23 antibody
and could explain the differences in the affinity values obtained by the different
measurement methods. Compared to the murine B1-23 antibody, the H1L5 humanized Bl-23
antibody surprisingly showed no tendency to aggregate (see also Example 9 below).
[0246] In the present assay, the chimeric B1-23 anti-GDF-15 antibody and the H1L5 humanized
B1-23 antibody exhibited affinities to human GDF-15 which were about 2-fold and 5-fold
higher, respectively, than the affinity of the murine B1-23 anti-GDF-15 antibody.
Thus, the chimeric B1-23 anti-GDF-15 antibody and the H1L5 humanized B1-23 antibody
are high affinity antibodies.
Example 9: Aggregation studies of the anti-GDF-15 antibodies
[0247] In order to test the aggregation properties of anti-GDF-15 antibodies, antibody samples
were shaken for 48 hours at room temperature in microcentrifuge tubes, and subsequently,
the tubes were visually analyzed for aggregated antibody precipitates.
[0248] It was observed that compared to the murine B1-23 antibody, the H1L5 humanized B1-23
antibody surprisingly showed no tendency to aggregate, even when the antibody was
only present in phosphate-buffered saline (PBS), and when no stabilizing proteins
such as BSA were present.
[0249] Moreover, in freeze/thaw and dilution experiments, it was observed that the H1L5
humanized B1-23 antibody did not aggregate during any of the dilution steps or freeze/thaw
cycles.
[0250] Additionally, the following experiment was carried out (see Figure 7):
5 mg of antibody (B1-23, chimeric B1-23 = "ChimB1-23", H1L5) were loaded on Proteus™
protein A columns, eluted and collected in a TRIS Buffer at physiological pH. After
elution, the quality of the purified antibodies was assessed by Coomassie Brilliant
Blue gel analysis. The concentration of the eluted antibodies was measured photometrically
as well as in a Bradford assay (Roti-Quant, Carl Roth, Karlsruhe, Germany). All 3
antibody solutions (B1-23, ChimB1-23, H1L5) showed similar concentrations and were
adjusted to 0.5 mg/ml. The antibodies were then 10fold concentrated via spin columns
(Centricon, MWCO 30). After this step, turbidity indicated the presence of precipitates
in the sample containing B1-23, whereas ChimB1-23 and H1L5 showed no signs of aggregation.
A11 concentrated eluates were then centrifuged for 5 min at 13000 rpm in order to
precipitate antibody aggregates. The remaining amount of soluble antibodies was finally
determined via Bradford assay from the supernatant.
[0251] These properties of the antibodies are expected to be advantageous for clinical formulation
of the antibodies.
G) Industrial Applicability
[0252] The antibodies, antigen-binding portions thereof, pharmaceutical compositions and
kits according to the present invention may be industrially manufactured and sold
as products for the claimed methods and uses (e.g. for treating cancer cachexia and
cancer), in accordance with known standards for the manufacture of pharmaceutical
products. Accordingly, the present invention is industrially applicable.
Preferred Embodiments
[0253]
- 1. A monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof, wherein the heavy chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 90% identical
thereto, and wherein the light chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 85% identical
thereto, wherein the constant domain of the heavy chain comprises the amino acid sequence
of SEQ ID No: 29, or an amino acid sequence at least 85%, preferably at least 90%,
more preferably at least 95% identical thereto, and wherein the constant domain of
the light chain comprises the amino acid sequence of SEQ ID No: 32, or an amino acid
sequence at least 85%, preferably at least 90%, more preferably at least 95% identical
thereto.
- 2. A monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof, wherein the binding is binding to a conformational or discontinuous
epitope on human GDF-15 comprised by the amino acid sequences of SEQ ID No: 25 and
SEQ ID No: 26, wherein the constant domain of the heavy chain comprises the amino
acid sequence of SEQ ID No: 29, or an amino acid sequence at least 85%, preferably
at least 90%, more preferably at least 95% identical thereto, and wherein the constant
domain of the light chain comprises the amino acid sequence of SEQ ID No: 32, or an
amino acid sequence at least 85%, preferably at least 90%, more preferably at least
95% identical thereto.
- 3. The antibody or an antigen-binding portion thereof of item 1 or 2, wherein the
constant domain of the heavy chain comprises the amino acid sequence of SEQ ID No:
29, or an amino acid sequence at least 98%, preferably at least 99% identical thereto,
and wherein the constant domain of the light chain comprises the amino acid sequence
of SEQ ID No: 32, or an amino acid sequence at least 98%, preferably at least 99%
identical thereto.
- 4. The antibody or an antigen-binding portion thereof of any of items 1 to 3, wherein
the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID
No: 29, and wherein the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32.
- 5. The antibody or antigen-binding portion thereof of any one of items 1-4, wherein
the antibody is a humanized antibody.
- 6. The antibody or antigen-binding portion thereof of item 5, wherein all of the variable
domains of the antibody are humanized variable domains.
- 7. The antibody or antigen-binding portion thereof of any one of items 1-6, wherein
the heavy chain variable domain comprises the amino acid sequence of SEQ ID No: 28,
or an amino acid sequence at least 90%, preferably at least 95%, more preferably at
least 98%, still more preferably at least 99% identical thereto, and wherein the light
chain variable domain comprises the amino acid sequence of SEQ ID No: 31, or an amino
acid sequence at least 90%, preferably at least 95%, more preferably at least 98%,
still more preferably at least 99% identical thereto.
- 8. The antibody or antigen-binding portion thereof of any one of items 1-7, wherein
the heavy chain variable domain comprises the amino acid sequence of SEQ ID No: 28,
and wherein the light chain variable domain comprises the amino acid sequence of SEQ
ID No: 31.
- 9. The antibody or antigen-binding portion thereof of any one of items 1-8, wherein
the heavy chain comprises the amino acid sequence of SEQ ID No: 27, and wherein the
light chain comprises the amino acid sequence of SEQ ID No: 30.
- 10.The antibody or antigen-binding portion thereof of any one of items 1-4, wherein
the heavy chain variable domain comprises the amino acid sequence of SEQ ID No: 34,
or an amino acid sequence at least 75%, more preferably at least 90%, more preferably
at least 95%, more preferably at least 98%, still more preferably at least 99% identical
thereto, and wherein the light chain variable domain comprises the amino acid sequence
of SEQ ID No: 37, or an amino acid sequence at least 80%, more preferably at least
90%, more preferably at least 95%, more preferably at least 98%, still more preferably
at least 99% identical thereto.
- 11.The antibody or antigen-binding portion thereof of item 10, wherein the heavy chain
variable domain comprises the amino acid sequence of SEQ ID No: 34, and wherein the
light chain variable domain comprises the amino acid sequence of SEQ ID No: 37.
- 12. The antibody or antigen-binding portion thereof of any one of items 1-11, wherein
the heavy chain variable domain comprises a CDR1 region comprising the amino acid
sequence of SEQ ID NO: 3 and a CDR2 region comprising the amino acid sequence of SEQ
ID NO: 4, and wherein the light chain variable domain comprises a CDR1 region comprising
the amino acid sequence of SEQ ID NO: 6 and a CDR2 region comprising the amino acid
sequence ser-ala-ser.
- 13.The antibody or antigen-binding portion thereof of any one of items 2-12, wherein
the heavy chain variable domain comprises a CDR3 region comprising the amino acid
sequence of SEQ ID NO: 5 or an amino acid sequence at least 90% identical thereto,
and wherein the light chain variable domain comprises a CDR3 region comprising the
amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 85% identical
thereto.
- 14.The antibody or antigen-binding portion thereof of any one of items 1 and 3-12,
wherein the binding is binding to a conformational or discontinuous epitope on human
GDF-15 that is comprised by the amino acid sequences of SEQ ID No: 25 and SEQ ID No:
26.
- 15.The antibody or antigen-binding portion thereof of any one of items 1-14, wherein
the antibody has a size of more than 100 kDa, preferably more than 110 kDa, more preferably
more than 120 kDa, still more preferably more than 130 kDa, and most preferably more
than 140 kDa.
- 16.The antibody or antigen-binding portion thereof of item 15, wherein the antibody
is a full-length antibody.
- 17.The antibody or antigen-binding portion thereof of item 16, wherein the antibody
is a full-length IgG antibody, preferably a full-length IgG1 antibody.
- 18.The antibody or antigen-binding portion thereof of any one of items 1 to 17, wherein
the antibody has an Fc portion which is capable of binding to the Fc receptor.
- 19.The antibody or antigen-binding portion thereof of any one of items 1 to 18, wherein
the human GDF-15 is recombinant human GDF-15 having the amino acid sequence represented
by SEQ ID No: 8.
- 20.An antibody or antigen-binding portion thereof according to any one of items 1
to 19 for use in medicine.
- 21.An antibody or antigen-binding portion thereof according to any one of items 1
to 19, for use in a method for treating cancer cachexia in a mammal.
- 22.An antibody or antigen-binding portion thereof according to any one of items 1
to 19, for use in a method for treating cancer in a mammal.
- 23.An antibody or antigen-binding portion thereof according to any one of items 21
to 22 for use according to any one of items 21 to 22, wherein the method is a method
for both treating cancer and treating cancer cachexia in the same mammal.
- 24.The antibody or antigen-binding portion thereof of any one of items 21 to 23 for
the use according to any one of items 21 to 23, wherein the mammal is a human patient.
- 25.The antibody or antigen-binding portion thereof of item 21 or 23-24 for the use
according to item 21 or 23-24, wherein the method for treating cancer cachexia is
a method for completely preventing or completely reverting cancer cachexia.
- 26.The antibody or antigen-binding portion thereof of item 25 for the use according
to item 25, wherein the method for treating cancer cachexia is a method for completely
preventing cancer cachexia.
- 27.The antibody or antigen-binding portion thereof of item 25 for the use according
to item 25, wherein the method for treating cancer cachexia is a method for completely
reverting cancer cachexia.
- 28.The antibody or antigen-binding portion thereof of any one of items 21-27 for the
use according to any one of items 21-27, wherein in the method, only mammals suffering
from both
- i) the cancer, and
- ii) cancer cachexia
are treated.
- 29.The antibody or antigen-binding portion thereof of any one of items 21 or 23-28
for the use according to any one of items 21 or 23-28, wherein the method increases
body weight of the mammal compared to its body weight before the onset of cancer cachexia.
- 30.The antibody or antigen-binding portion thereof of item 29 for the use according
to item 29, wherein the increase in body weight of the mammal is at least 1.5%, preferably
at least 2.5%, more preferably at least 5% compared to its body weight before the
onset of cancer cachexia.
- 31.The antibody or antigen-binding portion thereof of any one of items 21 to 30 for
the use according to any one of items 21 to 30, wherein the cancer cells of the mammal
endogenously express GDF-15 and/or the cancer cells of the mammal stimulate endogenous
expression of GDF-15 in non-cancerous cells of the mammal.
- 32.The antibody or antigen-binding portion thereof of any one of items 21 to 31 for
the use according to any one of items 21 to 31, wherein the cancer cells of the mammal
endogenously express GDF-15.
- 33.The antibody or antigen-binding portion thereof of any one of items 22-32 for the
use according to any one of items 22-32, wherein the method for treating cancer is
a method comprising inhibition of cancer growth.
- 34.The antibody or antigen-binding portion thereof of any one of items 22-33 for the
use according to any one of items 22-33, wherein the method for treating cancer comprises
the induction of killing of cancer cells by NK cells and CD8+ T cells in the human
patient.
- 35.The antibody or antigen-binding portion thereof of any one of items 21-34 for the
use according to any one of items 21-34, wherein the human patient has elevated GDF-15
levels in blood serum before administration.
- 36.The antibody or antigen-binding portion thereof of any one of items 21-35 for the
use according to any one of items 21-35, wherein the antibody or antigen-binding portion
thereof is
- A) the sole ingredient pharmaceutically active against cancer used in the method,
or
- B) used in combination with one or more further ingredients pharmaceutically active
against cancer.
- 37.The antibody or antigen-binding portion thereof of any one of items 21-36 for the
use according to any one of items 21-36, wherein the cancer is selected from the group
consisting of brain cancers including glioma, cancers of the nervous system, melanoma,
lung cancer, lip and oral cavity cancer, hepatic carcinoma, leukemia, Hodgkin lymphoma,
Non-Hodgkin lymphoma, bladder cancer, cervix uteri cancer, corpus uteri cancer, testis
cancer, thyroid cancer, kidney cancer, gallbladder cancer, multiple myeloma, nasopharynx
cancer, larynx cancer, pharynx cancer, oesophagus cancer, gastrointestinal tumors
including stomach and colorectal cancer, pancreatic cancer, prostate cancer, ovarian
cancer and breast cancer, preferably from the group consisting of melanoma, prostate
cancer, breast cancer, brain cancers including glioma, colorectal cancer, stomach
cancer, oesophagus cancer and ovarian cancer, and most preferably is melanoma.
- 38.The antibody or antigen-binding portion thereof of any one of items 21-37 for the
use according to any one of items 21-37, wherein prior to administration, the tumor
or tumors formed by the cancer have higher human GDF-15 levels compared to a control
sample of the same patient obtained from a non-cancerous part of the tissue which
is the tissue of origin of the cancer, preferably 1.2-fold higher levels, more preferably
1.5-fold higher levels, still more preferably 2-fold higher levels and most preferably
5-fold higher levels.
- 39.The antibody or antigen-binding portion thereof of item 38 for the use according
to item 38, wherein the antibody or antigen-binding portion thereof is used in combination
with one or more further ingredients pharmaceutically active against cancer, and wherein
the one or more further ingredients pharmaceutically active against cancer are selected
from the group consisting of: alkylating agents; anti-metabolites; alkaloids, taxanes;
topoisomerase inhibitors; cytotoxic antibiotics; and radioisotopes.
- 40.The antibody or antigen-binding portion thereof of item 39 for the use according
to item 39, wherein the one or more further ingredients pharmaceutically active against
cancer are selected from the group consisting of: cisplatin, carboplatin, oxaliplatin,
mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide; azathioprine and
mercaptopurine; vincristine, vinblastine, vinorelbine, and vindesine, paclitaxel,
docetaxel, etoposide and teniposide; irinotecan and topotecan; actinomycin, anthracyclines,
doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin
and mitomycin.
- 41.A kit comprising the antibody or antigen-binding portion thereof of any one of
items 1-19.
- 42.The kit of item 41 for a use according to any one of items 21 to 40.
- 43.An expression vector comprising a nucleotide sequence encoding the antibody or
antigen-binding portion thereof according to any of items 1-19.
- 44.A cell line capable of producing an antibody or antigen-binding portion thereof
according to any one of items 1 to 19.
- 45.A monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof, wherein the heavy chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 90% identical
thereto, and wherein the light chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 85% identical
thereto, for use in a method for treating cancer cachexia in a mammal.
- 46.A monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof, wherein the binding is binding to a conformational or discontinuous
epitope on human GDF-15 comprised by the amino acid sequences of SEQ ID No: 25 and
SEQ ID No: 26, for use in a method for treating cancer cachexia in a mammal.
- 47.The antibody or antigen-binding portion thereof of item 45 or 46 for the use according
to item 45 or 46, wherein the method for treating cancer cachexia is a method for
completely preventing or completely reverting cancer cachexia.
- 48.The antibody or antigen-binding portion thereof of item 47 for the use according
to item 47, wherein the method for treating cancer cachexia is a method for completely
preventing cancer cachexia.
- 49.The antibody or antigen-binding portion thereof of item 47 for the use according
to item 47, wherein the method for treating cancer cachexia is a method for completely
reverting cancer cachexia.
- 50.The antibody or antigen-binding portion thereof of any one of items 45-49 for the
use according to any one of items 45-49, wherein in the method, only mammals suffering
from both
iii) the cancer, and
iv) cancer cachexia
are treated.
- 51. The antibody or antigen-binding portion thereof of any one of items 49-50 for
the use according to any one of items 49-50, wherein the method increases body weight
of the mammal compared to its body weight before the onset of cancer cachexia.
- 52.The antibody or antigen-binding portion thereof of item 51 for the use according
to item 51, wherein the increase in body weight of the mammal is at least 1.5%, preferably
at least 2.5%, more preferably at least 5% compared to its body weight before the
onset of cancer cachexia.
- 53.The antibody or antigen-binding portion thereof of any one of items 45-52 for the
use according to any one of items 45-52, wherein the method is a method for both treating
cancer and treating cancer cachexia in the same mammal.
- 54.The antibody or antigen-binding portion thereof of any one of items 45-53 for the
use according to any one of items 45-53, wherein the antibody has a size of more than
100 kDa, preferably more than 110 kDa, more preferably more than 120 kDa, still more
preferably more than 130 kDa, and most preferably more than 140 kDa.
- 55.The antibody or antigen-binding portion thereof of item 54 for the use according
to item 54, wherein the antibody is a full-length antibody.
- 56.The antibody or antigen-binding portion thereof of item 55 for the use according
to item 55, wherein the antibody is a full-length IgG antibody.
- 57.The antibody or antigen-binding portion thereof of any one of items 45 to 56 for
the use according to any one of items 45 to 56, wherein the antibody has an Fc portion
which is capable of binding to the Fc receptor.
- 58.The antibody or antigen-binding portion thereof of any one of items 45 to 57 for
the use according to any one of items 45 to 57, wherein the cancer cells of the mammal
endogenously express GDF-15 and/or the cancer cells of the mammal stimulate endogenous
expression of GDF-15 in non-cancerous cells of the mammal.
- 59.The antibody or antigen-binding portion thereof of any one of items 45 to 58 for
the use according to any one of items 45 to 58, wherein the cancer cells of the mammal
endogenously express GDF-15.
- 60.The antibody or antigen-binding portion thereof of any one of items 45 to 59 for
the use according to any one of items 45 to 59, wherein the mammal is a human patient.
- 61.The antibody or antigen-binding portion thereof of any one of items 45 to 60 for
the use according to any one of items 45 to 60, wherein the human GDF-15 is recombinant
human GDF-15 having the amino acid sequence represented by SEQ ID No: 8.
- 62.The antibody or antigen-binding portion thereof of any one of items 46-61 for the
use according to any one of items 46-61, wherein the heavy chain variable domain comprises
a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid
sequence at least 90% identical thereto, and wherein the light chain variable domain
comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 7 or an amino
acid sequence at least 85% identical thereto.
- 63.The antibody or antigen-binding portion thereof of any one of items 45 and 47-61
for the use according to any one of items 45 and 47-61, wherein the binding is binding
to a conformational or discontinuous epitope on human GDF-15 that is comprised by
the amino acid sequences of SEQ ID No: 25 and SEQ ID No: 26.
- 64.The antibody or antigen-binding portion thereof of any one of items 53-63 for the
use according to any one of items 53-63, wherein the method for treating cancer is
a method comprising inhibition of cancer growth.
- 65.The antibody or antigen-binding portion thereof of any one of items 53-64 for the
use according to any one of items 53-64, wherein the method for treating cancer comprises
the induction of killing of cancer cells by NK cells and CD8+ T cells in the human
patient.
- 66.The antibody or antigen-binding portion thereof of any one of items 45-65 for the
use according to any one of items 45-65, wherein the heavy chain variable domain comprises
a CDR1 region comprising the amino acid sequence of SEQ ID NO: 3 and a CDR2 region
comprising the amino acid sequence of SEQ ID NO: 4, and wherein the light chain variable
domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 6
and a CDR2 region comprising the amino acid sequence ser-ala-ser.
- 67.The antibody or antigen-binding portion thereof of any one of items 45-66 for the
use according to any one of items 45-66, wherein the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95% identical thereto,
and wherein the constant domain of the light chain comprises the amino acid sequence
of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least 90%,
more preferably at least 95% identical thereto.
- 68.The antibody or antigen-binding portion thereof of any one of items 45-67 for the
use according to any one of items 45-67, wherein the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 98%, preferably at least 99% identical thereto, and wherein the constant
domain of the light chain comprises the amino acid sequence of SEQ ID No: 32, or an
amino acid sequence at least 98%, preferably at least 99% identical thereto.
- 69. The antibody or antigen-binding portion thereof of any one of items 45-68 for
the use according to any one of items 45-68, wherein the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, and wherein the constant
domain of the light chain comprises the amino acid sequence of SEQ ID No: 32.
- 70. The antibody or antigen-binding portion thereof of any one of items 45-69 for
the use according to any one of items 45-69, wherein the antibody is a humanized antibody,
and wherein all of the variable domains of the antibody are humanized variable domains.
- 71.The antibody or antigen-binding portion thereof of any one of items 45-70 for the
use according to any one of items 45-70, wherein the heavy chain variable domain comprises
the amino acid sequence of SEQ ID No: 28, or an amino acid sequence at least 90%,
preferably at least 95%, more preferably at least 98%, still more preferably at least
99% identical thereto, and wherein the light chain variable domain comprises the amino
acid sequence of SEQ ID No: 31, or an amino acid sequence at least 90%, preferably
at least 95%, more preferably at least 98%, still more preferably at least 99% identical
thereto.
- 72.The antibody or antigen-binding portion thereof of any one of items 45-71 for the
use according to any one of items 45-71, wherein the heavy chain variable domain comprises
the amino acid sequence of SEQ ID No: 28, and wherein the light chain variable domain
comprises the amino acid sequence of SEQ ID No: 31.
- 73.The antibody or antigen-binding portion thereof of any one of items 45-72 for the
use according to any one of items 45-72, wherein the heavy chain comprises the amino
acid sequence of SEQ ID No: 27, and wherein the light chain comprises the amino acid
sequence of SEQ ID No: 30.
- 74.The antibody or antigen-binding portion thereof of any one of items 45-69 for the
use according to any one of items 45-69, wherein the heavy chain variable domain comprises
the amino acid sequence of SEQ ID No: 34, or an amino acid sequence at least 75%,
more preferably at least 90%, more preferably at least 95%, more preferably at least
98%, still more preferably at least 99% identical thereto, and wherein the light chain
variable domain comprises the amino acid sequence of SEQ ID No: 37, or an amino acid
sequence at least 80%, more preferably at least 90%, more preferably at least 95%,
more preferably at least 98%, still more preferably at least 99% identical thereto.
- 75.The antibody or antigen-binding portion thereof of item 74 for the use according
to item 74, wherein the heavy chain variable domain comprises the amino acid sequence
of SEQ ID No: 34, and wherein the light chain variable domain comprises the amino
acid sequence of SEQ ID No: 37.
- 76. The antibody or antigen-binding portion thereof of any one of items 45-66 for
the use according to any one of items 45-66, wherein the antibody is the antibody
to human GDF-15 obtainable from the cell line B1-23 deposited with the Deutsche Sammlung
für Mikroorganismen und Zellkulturen GmbH (DMSZ) under the accession No. DSM ACC3142
or an antigen-binding portion thereof.
- 77.The antibody or antigen-binding portion thereof of any one of items 45-75 for the
use according to any one of items 45-75, wherein the heavy chain variable domain comprises
a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5, or wherein the light
chain variable domain comprises a CDR3 region comprising the amino acid sequence of
SEQ ID NO: 7.
- 78.The antibody or antigen-binding portion thereof of any one of items 45-75 and 77
for the use according to any one of items 45-75 and 77, wherein the heavy chain variable
domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5,
and wherein the light chain variable domain comprises a CDR3 region comprising the
amino acid sequence of SEQ ID NO: 7.
- 79.The antibody or antigen-binding portion thereof of any one of items 45-69 and 77-78
for the use according to any one of items 45-69 and 77-78, wherein the heavy chain
variable domain comprises a region comprising an FR1, a CDR1, an FR2, a CDR2 and an
FR3 region and comprising the amino acid sequence of SEQ ID NO: 1 or a sequence 95%
identical thereto, and wherein the light chain variable domain comprises a region
comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region and comprising the amino
acid sequence of SEQ ID NO: 2 or a sequence 95% identical thereto.
- 80.The antibody or antigen-binding portion thereof of any one of items 45-69 and 77-79
for the use according to any one of items 45-69 and 77-79, wherein the heavy chain
variable domain comprises a region comprising an FR1, a CDR1, an FR2, a CDR2 and an
FR3 region and comprising the amino acid sequence of SEQ ID NO: 1 or a sequence 98%
identical thereto, and wherein the light chain variable domain comprises a region
comprising an FR1, a CDR1, an FR2, a CDR2 and an FR3 region and comprising the amino
acid sequence of SEQ ID NO: 2 or a sequence 98% identical thereto.
- 81.The antibody or antigen-binding portion thereof of any one of items 45-80 for the
use according to any one of items 45-80, wherein the antibody or antigen-binding portion
thereof has an equilibrium dissociation constant for human GDF-15 that is equal to
or less than 20 nM, preferably less than 10 nM, more preferably less than 5 nM and
most preferably between 0.1 nM and 2 nM.
- 82.The antibody or antigen-binding portion thereof of any one of items 45-75 and 77-81
for the use according to any one of items 45-75 and 77-81, wherein the antibody or
antigen-binding portion thereof binds to the same human GDF-15 epitope as the antibody
to human GDF-15 obtainable from the cell line B1-23 deposited with the Deutsche Sammlung
für Mikroorganismen und Zellkulturen GmbH (DMSZ) under the accession No. DSM ACC3142.
- 83.The antibody or antigen-binding portion thereof of any one of items 45-82 for the
use according to any one of items 45-82, wherein the human patient has elevated GDF-15
levels in blood serum before administration.
- 84.The antibody or antigen-binding portion thereof of any one of items 45-83 for the
use according to any one of items 45-83, wherein the antibody or antigen-binding portion
thereof is
- A) the sole ingredient pharmaceutically active against cancer used in the method,
or
- B) used in combination with one or more further ingredients pharmaceutically active
against cancer.
- 85.The antibody or antigen-binding portion thereof of any one of items 45-84 for the
use according to any one of items 45-84, wherein the cancer is selected from the group
consisting of brain cancers including glioma, cancers of the nervous system, melanoma,
lung cancer, lip and oral cavity cancer, hepatic carcinoma, leukemia, Hodgkin lymphoma,
Non-Hodgkin lymphoma, bladder cancer, cervix uteri cancer, corpus uteri cancer, testis
cancer, thyroid cancer, kidney cancer, gallbladder cancer, multiple myeloma, nasopharynx
cancer, larynx cancer, pharynx cancer, oesophagus cancer, gastrointestinal tumors
including stomach and colorectal cancer, pancreatic cancer, prostate cancer, ovarian
cancer and breast cancer, preferably from the group consisting of melanoma, prostate
cancer, breast cancer, brain cancers including glioma, colorectal cancer, stomach
cancer, oesophagus cancer and ovarian cancer, and most preferably is melanoma.
- 86.The antibody or antigen-binding portion thereof of any one of items 45-85 for the
use according to any one of items 45-85, wherein prior to administration, the tumor
or tumors formed by the cancer have higher human GDF-15 levels compared to a control
sample of the same patient obtained from a non-cancerous part of the tissue which
is the tissue of origin of the cancer, preferably 1.2-fold higher levels, more preferably
1.5-fold higher levels, still more preferably 2-fold higher levels and most preferably
5-fold higher levels.
- 87.The antibody or antigen-binding portion thereof of item 84 for the use according
to item 84, wherein the antibody or antigen-binding portion thereof is used in combination
with one or more further ingredients pharmaceutically active against cancer, and wherein
the one or more further ingredients pharmaceutically active against cancer are selected
from the group consisting of: alkylating agents; anti-metabolites; alkaloids, taxanes;
topoisomerase inhibitors; cytotoxic antibiotics; and radioisotopes.
- 88.The antibody or antigen-binding portion thereof of item 87 for the use according
to item 87, wherein the one or more further ingredients pharmaceutically active against
cancer are selected from the group consisting of: cisplatin, carboplatin, oxaliplatin,
mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide; azathioprine and
mercaptopurine; vincristine, vinblastine, vinorelbine, and vindesine, paclitaxel,
docetaxel, etoposide and teniposide; irinotecan and topotecan; actinomycin, anthracyclines,
doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin
and mitomycin.
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WO 2005/099746
WO 2009/021293
[0255] The present invention also includes the following items.
- 1. A monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof, wherein the heavy chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 90% identical
thereto, and wherein the light chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 85% identical
thereto, wherein the constant domain of the heavy chain comprises the amino acid sequence
of SEQ ID No: 29, or an amino acid sequence at least 85% identical thereto, and wherein
the constant domain of the light chain comprises the amino acid sequence of SEQ ID
No: 32, or an amino acid sequence at least 85% identical thereto.
- 2. A monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof, wherein the binding is binding to a conformational or discontinuous
epitope on human GDF-15 comprised by the amino acid sequences of SEQ ID No: 25 and
SEQ ID No: 26, wherein the constant domain of the heavy chain comprises the amino
acid sequence of SEQ ID No: 29, or an amino acid sequence at least 85% identical thereto,
and wherein the constant domain of the light chain comprises the amino acid sequence
of SEQ ID No: 32, or an amino acid sequence at least 85% identical thereto.
- 3. The antibody or an antigen-binding portion thereof of item 1 or 2, wherein the
constant domain of the heavy chain comprises the amino acid sequence of SEQ ID No:
29, or an amino acid sequence at least 90%, more preferably at least 95% identical
thereto, and wherein the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 90%, more preferably
at least 95% identical thereto.
- 4. The antibody or an antigen-binding portion thereof of any one of items 1 to 3,
wherein the constant domain of the heavy chain comprises the amino acid sequence of
SEQ ID No: 29, or an amino acid sequence at least 98%, preferably at least 99% identical
thereto, and wherein the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32, or an amino acid sequence at least 98%, preferably at least
99% identical thereto.
- 5. The antibody or an antigen-binding portion thereof of any of items 1 to 4, wherein
the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID
No: 29, and wherein the constant domain of the light chain comprises the amino acid
sequence of SEQ ID No: 32.
- 6. The antibody or antigen-binding portion thereof of any one of items 1-5, wherein
the antibody is a humanized antibody.
- 7. The antibody or antigen-binding portion thereof of item 6, wherein all of the variable
domains of the antibody are humanized variable domains.
- 8. The antibody or antigen-binding portion thereof of any one of items 1-7, wherein
the heavy chain variable domain comprises the amino acid sequence of SEQ ID No: 28,
or an amino acid sequence at least 90%, preferably at least 95%, more preferably at
least 98%, still more preferably at least 99% identical thereto, and wherein the light
chain variable domain comprises the amino acid sequence of SEQ ID No: 31, or an amino
acid sequence at least 90%, preferably at least 95%, more preferably at least 98%,
still more preferably at least 99% identical thereto.
- 9. The antibody or antigen-binding portion thereof of any one of items 1-8, wherein
the heavy chain variable domain comprises the amino acid sequence of SEQ ID No: 28,
and wherein the light chain variable domain comprises the amino acid sequence of SEQ
ID No: 31.
- 10.The antibody or antigen-binding portion thereof of any one of items 1-9, wherein
the heavy chain comprises the amino acid sequence of SEQ ID No: 27, and wherein the
light chain comprises the amino acid sequence of SEQ ID No: 30.
- 11.The antibody or antigen-binding portion thereof of any one of items 1-5, wherein
the heavy chain variable domain comprises the amino acid sequence of SEQ ID No: 34,
or an amino acid sequence at least 75%, more preferably at least 90%, more preferably
at least 95%, more preferably at least 98%, still more preferably at least 99% identical
thereto, and wherein the light chain variable domain comprises the amino acid sequence
of SEQ ID No: 37, or an amino acid sequence at least 80%, more preferably at least
90%, more preferably at least 95%, more preferably at least 98%, still more preferably
at least 99% identical thereto.
- 12.The antibody or antigen-binding portion thereof of item 11, wherein the heavy chain
variable domain comprises the amino acid sequence of SEQ ID No: 34, and wherein the
light chain variable domain comprises the amino acid sequence of SEQ ID No: 37.
- 13. The antibody or antigen-binding portion thereof of any one of items 1-12, wherein
the heavy chain variable domain comprises a CDR1 region comprising the amino acid
sequence of SEQ ID NO: 3 and a CDR2 region comprising the amino acid sequence of SEQ
ID NO: 4, and wherein the light chain variable domain comprises a CDR1 region comprising
the amino acid sequence of SEQ ID NO: 6 and a CDR2 region comprising the amino acid
sequence ser-ala-ser.
- 14.The antibody or antigen-binding portion thereof of any one of items 2-13, wherein
the heavy chain variable domain comprises a CDR3 region comprising the amino acid
sequence of SEQ ID NO: 5 or an amino acid sequence at least 90% identical thereto,
and wherein the light chain variable domain comprises a CDR3 region comprising the
amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 85% identical
thereto.
- 15.The antibody or antigen-binding portion thereof of any one of items 1 and 3-13,
wherein the binding is binding to a conformational or discontinuous epitope on human
GDF-15 that is comprised by the amino acid sequences of SEQ ID No: 25 and SEQ ID No:
26.
- 16.The antibody or antigen-binding portion thereof of any one of items 1-15, wherein
the antibody has a size of more than 100 kDa, preferably more than 110 kDa, more preferably
more than 120 kDa, still more preferably more than 130 kDa, and most preferably more
than 140 kDa.
- 17.The antibody or antigen-binding portion thereof of item 16, wherein the antibody
is a full-length antibody.
- 18.The antibody or antigen-binding portion thereof of item 17, wherein the antibody
is a full-length IgG antibody, preferably a full-length IgG1 antibody.
- 19.The antibody or antigen-binding portion thereof of any one of items 1 to 18, wherein
the antibody has an Fc portion which is capable of binding to the Fc receptor.
- 20.The antibody or antigen-binding portion thereof of any one of items 1 to 19, wherein
the human GDF-15 is recombinant human GDF-15 having the amino acid sequence represented
by SEQ ID No: 8.
- 21.The antibody or antigen-binding portion thereof according to any one of items 1
to 20 for use in medicine.
- 22.An antibody or antigen-binding portion thereof according to any one of items 1
to 20, for use in a method for treating cancer cachexia in a mammal.
- 23.An antibody or antigen-binding portion thereof according to any one of items 1
to 20, for use in a method for treating cancer in a mammal.
- 24.An antibody or antigen-binding portion thereof according to any one of items 22
to 23 for use according to any one of items 22 to 23, wherein the method is a method
for both treating cancer and treating cancer cachexia in the same mammal.
- 25.The antibody or antigen-binding portion thereof of any one of items 22 to 24 for
the use according to any one of items 22 to 24, wherein the mammal is a human patient.
- 26.A kit comprising the antibody or antigen-binding portion thereof of any of items
1-20.
- 27.An expression vector comprising a nucleotide sequence encoding the antibody or
antigen-binding portion thereof according to any of items 1-20.
- 28.A cell line capable of producing an antibody or antigen-binding portion thereof
according to any one of items 1 to 20.
- 29.A monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof, wherein the heavy chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 90% identical
thereto, and wherein the light chain variable domain comprises a CDR3 region comprising
the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 85% identical
thereto, for use in a method for treating cancer cachexia in a mammal.
- 30.A monoclonal antibody capable of binding to human GDF-15, or an antigen-binding
portion thereof, wherein the binding is binding to a conformational or discontinuous
epitope on human GDF-15 comprised by the amino acid sequences of SEQ ID No: 25 and
SEQ ID No: 26, for use in a method for treating cancer cachexia in a mammal.
- 31.The antibody or antigen-binding portion thereof of item 29 or 30 for the use according
to item 29 or 30, wherein the method for treating cancer cachexia is a method for
completely preventing or completely reverting cancer cachexia.
- 32.The antibody or antigen-binding portion thereof of item 31 for the use according
to item 31, wherein the method for treating cancer cachexia is a method for completely
preventing cancer cachexia.
- 33.The antibody or antigen-binding portion thereof of item 31 for the use according
to item 31, wherein the method for treating cancer cachexia is a method for completely
reverting cancer cachexia.
- 34.The antibody or antigen-binding portion thereof of any one of items 29-33 for the
use according to any one of items 29-33, wherein in the method, only mammals suffering
from both
- i) the cancer, and
- ii) cancer cachexia
are treated.
- 35.The antibody or antigen-binding portion thereof of any one of items 33-34 for the
use according to any one of items 33-34, wherein the method increases body weight
of the mammal compared to its body weight before the onset of cancer cachexia.
- 36.The antibody or antigen-binding portion thereof of item 35 for the use according
to item 35, wherein the increase in body weight of the mammal is at least 1.5%, preferably
at least 2.5%, more preferably at least 5% compared to its body weight before the
onset of cancer cachexia.
- 37.The antibody or antigen-binding portion thereof of any one of items 29-36 for the
use according to any one of items 29-36, wherein the method is a method for both treating
cancer and treating cancer cachexia in the same mammal.
- 38.The antibody or antigen-binding portion thereof of any one of items 29-37 for the
use according to any one of items 29-37, wherein the antibody has a size of more than
100 kDa, preferably more than 110 kDa, more preferably more than 120 kDa, still more
preferably more than 130 kDa, and most preferably more than 140 kDa.
- 39.The antibody or antigen-binding portion thereof of item 38 for the use according
to item 38, wherein the antibody is a full-length antibody.
- 40.The antibody or antigen-binding portion thereof of item 39 for the use according
to item 39, wherein the antibody is a full-length IgG antibody.
- 41.The antibody or antigen-binding portion thereof of any one of items 29 to 40 for
the use according to any one of items 29 to 40, wherein the antibody has an Fc portion
which is capable of binding to the Fc receptor.
- 42.The antibody or antigen-binding portion thereof of any one of items 29 to 41 for
the use according to any one of items 29 to 41, wherein the cancer cells of the mammal
endogenously express GDF-15 and/or the cancer cells of the mammal stimulate endogenous
expression of GDF-15 in non-cancerous cells of the mammal.
- 43.The antibody or antigen-binding portion thereof of any one of items 29 to 42 for
the use according to any one of items 29 to 42, wherein the cancer cells of the mammal
endogenously express GDF-15.
- 44.The antibody or antigen-binding portion thereof of any one of items 29 to 43 for
the use according to any one of items 29 to 43, wherein the mammal is a human patient.
- 45.The antibody or antigen-binding portion thereof of any one of items 29 to 44 for
the use according to any one of items 29 to 44, wherein the human GDF-15 is recombinant
human GDF-15 having the amino acid sequence represented by SEQ ID No: 8.
- 46.The antibody or antigen-binding portion thereof of any one of items 30-45 for the
use according to any one of items 30-45, wherein the heavy chain variable domain comprises
a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid
sequence at least 90% identical thereto, and wherein the light chain variable domain
comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 7 or an amino
acid sequence at least 85% identical thereto.
- 47.The antibody or antigen-binding portion thereof of any one of items 29 and 31-45
for the use according to any one of items 29 and 31-45, wherein the binding is binding
to a conformational or discontinuous epitope on human GDF-15 that is comprised by
the amino acid sequences of SEQ ID No: 25 and SEQ ID No: 26.
- 48.The antibody or antigen-binding portion thereof of any one of items 37-47 for the
use according to any one of items 37-47, wherein the method for treating cancer is
a method comprising inhibition of cancer growth.
- 49.The antibody or antigen-binding portion thereof of any one of items 37-48 for the
use according to any one of items 37-48, wherein the method for treating cancer comprises
the induction of killing of cancer cells by NK cells and CD8+ T cells in the human
patient.
- 50.The antibody or antigen-binding portion thereof of any one of items 29-49 for the
use according to any one of items 29-49, wherein the heavy chain variable domain comprises
a CDR1 region comprising the amino acid sequence of SEQ ID NO: 3 and a CDR2 region
comprising the amino acid sequence of SEQ ID NO: 4, and wherein the light chain variable
domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 6
and a CDR2 region comprising the amino acid sequence ser-ala-ser.
- 51.The antibody or antigen-binding portion thereof of any one of items 29-50 for the
use according to any one of items 29-50, wherein the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 85%, preferably at least 90%, more preferably at least 95% identical thereto,
and wherein the constant domain of the light chain comprises the amino acid sequence
of SEQ ID No: 32, or an amino acid sequence at least 85%, preferably at least 90%,
more preferably at least 95% identical thereto.
- 52.The antibody or antigen-binding portion thereof of any one of items 29-51 for the
use according to any one of items 29-51, wherein the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, or an amino acid sequence
at least 98%, preferably at least 99% identical thereto, and wherein the constant
domain of the light chain comprises the amino acid sequence of SEQ ID No: 32, or an
amino acid sequence at least 98%, preferably at least 99% identical thereto.
- 53. The antibody or antigen-binding portion thereof of any one of items 29-52 for
the use according to any one of items 29-52, wherein the constant domain of the heavy
chain comprises the amino acid sequence of SEQ ID No: 29, and wherein the constant
domain of the light chain comprises the amino acid sequence of SEQ ID No: 32.
- 54. The antibody or antigen-binding portion thereof of any one of items 29-53 for
the use according to any one of items 29-53, wherein the antibody is a humanized antibody,
and wherein all of the variable domains of the antibody are humanized variable domains.
- 55.The antibody or antigen-binding portion thereof of any one of items 29-54 for the
use according to any one of items 29-54, wherein the heavy chain variable domain comprises
the amino acid sequence of SEQ ID No: 28, or an amino acid sequence at least 90%,
preferably at least 95%, more preferably at least 98%, still more preferably at least
99% identical thereto, and wherein the light chain variable domain comprises the amino
acid sequence of SEQ ID No: 31, or an amino acid sequence at least 90%, preferably
at least 95%, more preferably at least 98%, still more preferably at least 99% identical
thereto.
- 56.The antibody or antigen-binding portion thereof of any one of items 29-55 for the
use according to any one of items 29-55, wherein the heavy chain variable domain comprises
the amino acid sequence of SEQ ID No: 28, and wherein the light chain variable domain
comprises the amino acid sequence of SEQ ID No: 31.
- 57.The antibody or antigen-binding portion thereof of any one of items 29-56 for the
use according to any one of items 29-56, wherein the heavy chain comprises the amino
acid sequence of SEQ ID No: 27, and wherein the light chain comprises the amino acid
sequence of SEQ ID No: 30.
- 58.The antibody or antigen-binding portion thereof of any one of items 29-53 for the
use according to any one of items 29-53, wherein the heavy chain variable domain comprises
the amino acid sequence of SEQ ID No: 34, or an amino acid sequence at least 75%,
more preferably at least 90%, more preferably at least 95%, more preferably at least
98%, still more preferably at least 99% identical thereto, and wherein the light chain
variable domain comprises the amino acid sequence of SEQ ID No: 37, or an amino acid
sequence at least 80%, more preferably at least 90%, more preferably at least 95%,
more preferably at least 98%, still more preferably at least 99% identical thereto.
- 59.The antibody or antigen-binding portion thereof of item 58 for the use according
to item 58, wherein the heavy chain variable domain comprises the amino acid sequence
of SEQ ID No: 34, and wherein the light chain variable domain comprises the amino
acid sequence of SEQ ID No: 37.
- 60. The antibody or antigen-binding portion thereof of any one of items 29-50 for
the use according to any one of items 29-50, wherein the antibody is the antibody
to human GDF-15 obtainable from the cell line Bl-23 deposited with the Deutsche Sammlung
für Mikroorganismen und Zellkulturen GmbH (DMSZ) under the accession No. DSM ACC3142
or an antigen-binding portion thereof.
